Method for quantitatively determining activity of protein-L-isoaspartic acid (D-aspartic acid)-methyltransferase and application of protein-L-isoaspartic acid (D-aspartic acid)-methyltransferase

Abstract

The invention provides a method for quantitatively determining the activity of protein-L-isoaspartic acid (D-aspartic acid)-methyltransferase and application of the method. The method comprises the following steps: transferring methyl on S-adenosylmethionine (SAM) to a polypeptide substrate through PCMT1 to generate S-adenosylhomocysteine (SAH), hydrolyzing into homocysteine under the catalysis of S-adenosylhomocysteine hydrolase (SAHH), carrying out quantitative Michael addition reaction with a fluorescent dye, and quantitatively determining the activity of PCMT1 by detecting the change of a fluorescence signal of a product. The invention further provides application of the method for quantitatively determining the activity of the protein-L-isoaspartic acid (D-aspartic acid)-methyltransferase (PCMT), a large number of PCMT1 inhibitors can be rapidly and efficiently screened out through the method, and an important tool is provided for developing lead compounds for cancer prevention and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a method for quantitatively determining the activity of protein-L-isaspartate (D-aspartate)-methyltransferase and its application. Background Art

[0002] Aspartic acid (Asp) and aspartyl residues undergo a series of spontaneous damage reactions during the aging process, including deamidation, isomerization, and racemization. These reactions lead to partial destruction of the protein structure and function. With the accumulation of damage, it may ultimately lead to the loss of cell function, and further cause aging and the occurrence of related diseases. Protein-L-isaspartate (D-aspartate)-methyltransferase (PCMT1) plays a key repair role in this process. It can recognize and repair damaged L-isaspartate (L-isoAsp) residues, thereby effectively regulating protein function.

[0003] PCMT1 specifically recognizes the damaged forms of L-isoAsp and D-Asp residues, and initiates the repair cycle by transferring the methyl group on S-adenosylmethionine (SAM) to the side-chain carboxyl group of the L-isoAsp residue. In the repair reaction, the methyl ester spontaneously demethylates to generate cyclic L-succinimide, and this succinimide hydrolyzes to produce a mixture of aspartic acid (Asp) and isoaspartic acid (isoAsp) due to its instability. The continuous action of PCMT1 maintains the balance of the reaction, promotes the generation of L-aspartic acid, thereby achieving protein repair and restoring its structure and biological function.

[0004] Since the discovery of the catalytic activity of PCMT1, proteomics-based studies have identified approximately 30 endogenous substrates of PCMT1. These substrates are involved in a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The identification of related proteins provides a basis for further exploring the biological functions of PCMT1 in cells and in vivo. The wide distribution of PCMT1 in different tissues and its diverse substrates make it likely to participate in multiple biochemical pathways and cellular processes.

[0005] Latest research shows that PCMT1 plays a crucial role in aging, immune regulation, tumorigenesis, cell migration, invasion, apoptosis, and growth signaling pathways. In particular, PCMT1, which is overexpressed in various tumor cells, has been found to be closely related to the occurrence and development of cancer. Therefore, PCMT1 has become a potential cancer treatment target. Given the important role of PCMT1 in various physiological and pathological processes, the development of highly efficient and selective PCMT1 inhibitors has become a current research hotspot.

[0006] To study the biological function of PCMT1 and explore its potential therapeutic value, it is particularly important to establish a highly efficient and scalable PCMT1 activity detection method and a high-throughput inhibitor screening system. Through this system, not only can the accurate measurement of PCMT1 activity be achieved, but also effective inhibitors can be quickly screened out, providing new strategies for targeted therapy of cancer and other related diseases.

[0007] Currently, the detection methods for methyltransferase activity mainly include radiochemical method, high-performance liquid chromatography (HPLC) method, and fluorescence coupling method. In radiochemical analysis, radioactively labeled S-adenosylmethionine (SAM) is used as the methyl donor. When the isoAsp residue in the substrate is labeled and methylated by the catalysis of PCMT1, radioactively labeled methanol (MeOH) is released, and then measured using a scintillation counter. However, the main drawback of this method is the need to use radioactive compounds, which poses safety hazards and handling difficulties. The HPLC method uses a C18 reversed-phase chromatographic column to separate the methylation reaction product S-adenosylhomocysteine (SAH) and its substrate SAM, and quantitative analysis is carried out by ultraviolet detection. Although this method has high sensitivity and accuracy, its process is discontinuous and costly, and it is not suitable for high-throughput applications. The fluorescence coupling method establishes a coupling detection system based on PCMT1 and caspase-3. PCMT1 is a key enzyme for metabolizing isoaspartic acid peptides and proteins, and this system uses a fluorescent peptide probe containing an isoaspartic acid residue. After PCMT1 methylates the probe, the product is cleaved by caspase-3, activating the fluorescence signal. Although this method has high sensitivity, the synthesis of the probe is difficult and costly, limiting its application in large-scale high-throughput screening.

[0008] Therefore, there is an urgent need to develop a PCMT1 activity detection method with high sensitivity, simple quantification, low cost, and suitable for high-throughput screening, which is of crucial significance for promoting the functional research of PCMT1, drug screening, and targeted therapy of related diseases. Summary of the Invention

[0009] The present invention provides a method for quantitatively determining the activity of protein-L-is aspartic acid (D-aspartic acid)-methyltransferase and its application, aiming to solve the above problems existing in the background technology.

[0010] To achieve the above object, an embodiment of the present invention provides a method for quantitatively determining the activity of protein-L-is aspartic acid (D-aspartic acid)-methyltransferase and its application. The present invention uses SAH hydrolase to hydrolyze SAH into adenosine and homocysteine that have no inhibitory effect on PCMT1. Homocysteine undergoes a Michael addition reaction with a fluorescent dye, and by detecting the fluorescence intensity of the addition product at a specific excitation wavelength, the activity of PCMT1 is indirectly reflected. The present invention effectively eliminates the reverse inhibitory effect of SAH by skillfully coupling the three reaction steps of methylation transfer, SAH hydrolysis, and Michael addition, expands the linear range of the test, and extends the monitoring time.

[0011] An embodiment of the present invention provides a method for quantitatively determining the activity of protein-L-is aspartic acid (D-aspartic acid)-methyltransferase (PCMT1). This method is for non-disease diagnosis and treatment purposes and is characterized in that this method transfers the methyl group on S-adenosylmethionine (SAM) to a polypeptide substrate through PCMT1 to generate S-adenosylhomocysteine (SAH), which is hydrolyzed into homocysteine under the catalysis of S-adenosylhomocysteine hydrolase (SAHH), and undergoes a quantitative Michael addition reaction with a fluorescent dye. By detecting the change in the fluorescence signal of the product, the activity of PCMT1 is quantitatively determined;

[0012] The general structural formula of the polypeptide substrate is Wherein, R 1 , R 2 is selected from at least one of hydrophilic amino acids, hydrophobic amino acids, amino acids with charged groups, methylated or phosphorylated amino acids, fatty acylated amino acids, and cyanated amino acids. R 1 , R 2 can be a single amino acid residue or a combination of multiple amino acids to form a polypeptide chain, and can contain any type of modification or functional group, such as phosphate groups, sugar groups, alkyl groups, etc., as needed to enhance its interaction with PCMT1 or optimize the detection performance.

[0013] Preferably, the method includes the following steps:

[0014] S1. In a Tris-HCl buffer system with a pH value of 5.5 to 10.5, SAM and the polypeptide substrate are introduced and catalyzed by PCMT1. The incubation temperature for the enzyme-catalyzed reaction is 15 to 50 °C, and the time is 1 to 120 minutes to generate SAH; the concentration range of the polypeptide substrate and SAM is 0.1 to 10K m; The buffer solution is selected from 20 mM Tris-HCl, 10 mM Tris-HCl, 50 mM TrisHCl, 150 mM NaCl, etc.; More preferably, the incubation time for the enzymatic reaction is 1 to 60 minutes, and the incubation temperature range is 20 to 40 °C.

[0015] S2. Add a hydrolase that reacts with it to SAH for hydrolysis to generate homocysteine; the concentration range of the hydrolase is 0.1 to 500 μM; preferably, the concentration range is 0.1 to 50 μM;

[0016] S3. Add homocysteine to a fluorescent dye for reaction to generate a fluorescent product; the concentration range of the fluorescent dye is 0.1 to 500 μM; preferably, the concentration range is 0.1 to 50 μM;

[0017] S4. The activity of PCMT1 can be quantitatively determined by detecting the change in the fluorescent signal.

[0018] The analysis of the fluorescent signal is completed by a specific signal processing algorithm, including but not limited to: calculating the enzyme activity using the dynamic change rate of the fluorescent signal intensity. The change in the fluorescent signal can be a change in fluorescence intensity, fluorescence lifetime, or fluorescence polarization.

[0019] The system can be combined with an automated high-throughput screening platform to achieve efficient reaction condition control and fluorescent signal detection through microplates, multi-channel pipettes, or microfluidic devices.

[0020] Preferably, the structural general formula of the fluorescent dye is: Wherein, R 1 is a hydroxyl group, a straight-chain or branched-chain alkyl group with 1 to 10 carbon atoms, an aryl group, a halogenated alkyl group, or a heterocyclic group containing oxygen, nitrogen, or sulfur; R 5 is an ester group, an amide group, a cyano group, a sulfonamide group, a sulfonate group, or a derivative thereof; R 2 , R 3 and R 4 are selected from hydrogen, alkyl, aryl, halogen, hydroxyl, methoxy, nitro, amino. The fluorescent dye may also contain a hydrophilic group (such as a sulfonic acid group) or a hydrophobic group (such as a long-chain alkyl group) to enhance its solubility and stability in different systems.

[0021] Preferably, the polypeptide substrate is selected from at least one of KQVV-isoD-SAYEVIK, A-isoD-QLTEEQIAEFK, TS-isoD-TSKY, VYP-isoD-HA, VYP-isoD-DA, KASA-isoD-LAKY, VYP-isoD-AA, VYP-isoD-CA, VYP-isoD-SA, VYP-isoD-RR. The sequence of the polypeptide substrate can be other polypeptides containing the isoD site. The polypeptide substrate can also be chemically modified, fluorescently labeled or structurally optimized to improve the detection sensitivity or specificity. The modifications include but are not limited to:

[0022] a. Introducing fluorescent groups at both ends of the polypeptide sequence;

[0023] b. Optimizing the polypeptide sequence containing the isoD site to enhance the specific binding to PCMT1;

[0024] c. Improving the polypeptide stability through PEGylation or other modifications;

[0025] The polypeptide substrate can be obtained by classical solid-phase synthesis methods.

[0026] Preferably, the system further includes at least one of a surfactant and an anti-interference substance.

[0027] Among them, the surfactant is used to enhance the stability of the reaction system or improve the reaction efficiency, including but not limited to non-ionic, anionic, cationic or zwitterionic surfactants, such as Triton X-100, EDTA, emulsifier OP, Tween 20, Tween 80, etc., and its concentration range is 0.001% - 0.5%, preferably the concentration range is 0.01% - 0.1%.

[0028] The anti-interference substance is used to reduce the interference of non-specific reactions, including but not limited to thiol compounds, chelating agents or protein stabilizers.

[0029] Preferably, the S-adenosylhomocysteine hydrolase (SAHH) can be selected from wild-type and mutant SAHH with catalytic activity from any species source. Such as SAHH from mammals (such as humans, mice, rats, monkeys, etc.), birds, amphibians, reptiles, plants, fungi, bacteria and prokaryotes, wild-type SAHH with catalytic activity, mutant SAHH with catalytic activity, SAHH variants. The mutant SAHH may have characteristics such as an altered amino acid sequence, enhanced or weakened catalytic activity, different substrate specificities, etc.

[0030] Preferably, the PCMT1 can be selected from wild-type and mutant PCMT1 with catalytic activity in bacteria, insect cells, mammalian cells and yeast clone expression systems. Such as PCMT1 of mammals (such as humans, mice, rats, monkeys, etc.), birds, amphibians, reptiles, plants, fungi, bacteria and prokaryotes, wild-type PCMT1 with catalytic activity, mutant PCMT1 with catalytic activity, PCMT1 variants. Mutant PCMT1 may have characteristics such as altered amino acid sequence, enhanced or weakened catalytic activity, different substrate specificity, etc.

[0031] Preferably, the hydrophilic amino acid is selected from any one of histidine, alanine, glycine, serine, aspartic acid, and glutamic acid; the hydrophobic amino acid is selected from any one of cysteine, phenylalanine, tryptophan, isoleucine, valine, and leucine; and the amino acid with a charged group is selected from any one of lysine, arginine, aspartic acid, and glutamic acid.

[0032] The embodiments of the present invention also provide the application of the above method for quantitatively determining the activity of protein-L-isoaspartate (D-aspartate)-methyltransferase (PCMT1).

[0033] Preferably, the method can quickly and efficiently screen out a large number of PCMT1 inhibitors.

[0034] mechanism

[0035] PCMT1 transfers the methyl group on S-adenosylmethionine (SAM) to the polypeptide substrate to generate S-adenosylhomocysteine ​​(SAH). SAH is hydrolyzed to homocysteine ​​under the catalysis of S-adenosylhomocysteine ​​hydrolase (SAHH), and the latter undergoes a quantitative Michael addition reaction with a fluorescent dye to generate a product with a changed fluorescence characteristic. The activity of PCMT1 is quantitatively determined by detecting the change in the fluorescence intensity of the product.

[0036] The above scheme of the present invention has the following beneficial effects:

[0037] The SAH used in the present invention is one of the products of the PCMT1 methylation reaction and has strong reverse inhibitory activity, which will interfere with the inhibitory activity test. In order to eliminate the interference of SAH in the test, the present invention uses SAH hydrolase to hydrolyze SAH into adenosine and homocysteine ​​that have no inhibitory effect on PCMT1. Homocysteine ​​and fluorescent dye undergo a Mc-Gram addition reaction, and the activity of PCMT1 is indirectly reflected by detecting the fluorescence intensity of the addition product at a specific excitation wavelength. The present invention effectively eliminates the reverse inhibitory effect of SAH by cleverly coupling the three reaction steps of methyl transfer, SAH hydrolysis and Mc-Gram addition, expands the linear range of the test and prolongs the time available for monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0039] Figure 1 It is the SDS-PAGE detection result of preparing PCMT1 protein in the present invention; the lanes in the figure are UI (uninitiated), I (initiated), Sup (supernatant), Ppt (precipitate), Ub (waste liquid collected during loading), W (waste liquid flushed with Buffer A), Elu (target protein), Marker.

[0040] Figure 2 It is the SDS-PAGE detection result of preparing SAHH in the present invention. The lanes in the figure are UI (uninitiated), I (initiated), Sup (supernatant), Ppt (precipitate), Ub (waste liquid collected during loading), W (waste liquid flushed with Buffer A), Elu (target protein), Marker.

[0041] Figure 3 It is the schematic diagram of solid-phase synthesis of polypeptide substrate in the embodiment of the present invention.

[0042] Figure 4 It is the evaluation diagram of the influence of buffer solution and surfactant on the enzyme activity of PCMT1 in the embodiment of the present invention; A: Tris, B: Tris + trition.

[0043] Figure 5 It is the evaluation diagram of the influence of temperature on the enzymatic reaction activity of PCMT1 in the embodiment of the present invention.

[0044] Figure 6 It is the reaction linear range diagram for testing the concentration of PCMT1 in the embodiment of the present invention.

[0045] Figure 7 It is the influence of testing the concentrations of ThioGlo4 (A) and DMSO (B) on the reaction curve in the embodiment of the present invention.

[0046] Figure 8 It is the K m value.

[0047] Figure 9 It is the K m value of the polypeptide substrate VYP-isoD-HA in the embodiment of the present invention.

[0048] Figure 10Structural formulae of SAM, SAH, VYP-isoD-HA and schematic diagram of reaction mechanism according to embodiments of the present invention; A: Structural formulae of SAM, SAH, VYP-isoD-HA, B: Schematic diagram of reaction mechanism. Detailed implementation manners

[0049] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments. Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0050] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. The reagents and instruments used in the embodiments are all conventional selections in the art unless otherwise specified. The experimental methods without specific conditions noted in the embodiments are carried out under conventional conditions, such as the conditions described in the literature, books or the methods recommended by the manufacturers.

[0051] In view of the existing problems, embodiments of the present invention provide a method for quantitatively determining the activity of protein-L-isaspartate (D-aspartate)-methyltransferase (PCMT1). This method is for non-disease diagnosis and treatment purposes and is characterized in that the method transfers the methyl group on S-adenosylmethionine (SAM) to a polypeptide substrate through PCMT1 to generate S-adenosylhomocysteine (SAH), which is hydrolyzed to homocysteine under the catalysis of S-adenosylhomocysteine hydrolase (SAHH), and undergoes a quantitative Michael addition reaction with a fluorescent dye. By detecting the change in the fluorescence signal of the product, the activity of PCMT1 is quantitatively determined;

[0052] The general structural formula of the polypeptide substrate is wherein, R 1 , R 2 is selected from at least one of hydrophilic amino acids, hydrophobic amino acids, amino acids with charged groups, methylated or phosphorylated amino acids, fatty acylated amino acids, and cyanated amino acids. R 1 , R 2 can be a single amino acid residue or a combination of multiple amino acids to form a polypeptide chain, and can contain any type of modification or functional group as needed, such as phosphate groups, sugar groups, alkyl groups, etc., to enhance its interaction with PCMT1 or optimize the detection performance.

[0053] Preferably, the method includes the following steps:

[0054] S1. In a Tris-HCl buffer system with a pH value of 5.5 - 10.5, SAM and a polypeptide substrate are introduced and catalyzed by PCMT1. The incubation temperature for the enzymatic reaction is 15 - 50 °C and the time is 1 - 120 minutes to generate SAH; the concentration range of the polypeptide substrate and SAM is 0.1 - 10K m ; the buffer is selected from 20 mM Tris-HCl, 10 mM Tris-HCl, 50 mM TrisHCl, and 150 mM NaCl, etc.; more preferably, the incubation time for the enzymatic reaction is 1 - 60 minutes and the incubation temperature range is 20 - 40 °C.

[0055] S2. A hydrolase that reacts with it is added to SAH for hydrolysis to generate homocysteine; the concentration range of the hydrolase is 0.1 - 500 μM; preferably, the concentration range is 0.1 - 50 μM;

[0056] S3. Homocysteine is added to a fluorescent dye for reaction to generate a fluorescent product; the concentration range of the fluorescent dye is 0.1 - 500 μM; preferably, the concentration range is 0.1 - 50 μM;

[0057] S4. The activity of PCMT1 can be quantitatively determined by detecting the change in the fluorescent signal.

[0058] The analysis of the fluorescent signal is completed through a specific signal processing algorithm, including but not limited to: calculating the enzyme activity using the dynamic change rate of the fluorescent signal intensity. The change in the fluorescent signal can be a change in fluorescence intensity, fluorescence lifetime, or fluorescence polarization.

[0059] The system can be combined with an automated high-throughput screening platform to achieve efficient reaction condition control and fluorescent signal detection through microplates, multi-channel pipettes, or microfluidic devices.

[0060] Preferably, the general structural formula of the fluorescent dye is: wherein, R 1 is a hydroxyl group, a straight-chain or branched-chain alkyl group with 1 to 10 carbon atoms, an aryl group, a halogenated alkyl group, or a heterocyclic group containing oxygen, nitrogen, or sulfur; R 5 is an ester group, an amide, a cyano group, a sulfonamide group, a sulfonate group, or a derivative thereof; R 2 , R 3 and R 4 are selected from hydrogen, an alkyl group, an aryl group, a halogen, a hydroxyl group, a methoxy group, a nitro group, and an amino group. The fluorescent dye may also contain a hydrophilic group (such as a sulfonic acid group) or a hydrophobic group (such as a long-chain alkyl group) to enhance solubility and stability in different systems.

[0061] Preferably, the polypeptide substrate is selected from at least one of KQVV-isoD-SAYEVIK, A-isoD-QLTEEQIAEFK, TS-isoD-TSKY, VYP-isoD-HA, VYP-isoD-DA, KASA-isoD-LAKY, VYP-isoD-AA, VYP-isoD-CA, VYP-isoD-SA, VYP-isoD-RR. The sequence of the polypeptide substrate can be other polypeptides containing the isoD site. The polypeptide substrate can also be chemically modified, fluorescently labeled or structurally optimized to improve the detection sensitivity or specificity. The modifications include but are not limited to:

[0062] a. Introduce fluorescent groups at both ends of the polypeptide sequence;

[0063] b. Optimize the polypeptide sequence containing the isoD site to enhance specific binding to PCMT1;

[0064] c. Improve the polypeptide stability through PEGylation or other modifications;

[0065] The polypeptide substrate can be obtained by classical solid-phase synthesis methods.

[0066] Preferably, the system further includes at least one of a surfactant and an anti-interference substance.

[0067] Among them, the surfactant is used to enhance the stability of the reaction system or improve the reaction efficiency, including but not limited to non-ionic, anionic, cationic or zwitterionic surfactants, such as Triton X-100, EDTA, emulsifier OP, Tween 20, Tween 80, etc., and its concentration range is 0.001% - 0.5%, preferably the concentration range is 0.01% - 0.1%.

[0068] The anti-interference substance is used to reduce the interference of non-specific reactions, including but not limited to thiol compounds, chelating agents or protein stabilizers.

[0069] Preferably, the S-adenosylhomocysteine hydrolase (SAHH) can be selected from wild-type and mutant SAHHs with catalytic activity from any species source. Such as SAHHs from mammals (such as humans, mice, rats, monkeys, etc.), birds, amphibians, reptiles, plants, fungi, bacteria and prokaryotes, wild-type SAHHs with catalytic activity, mutant SAHHs with catalytic activity, SAHH variants. The mutant SAHH may have characteristics such as an altered amino acid sequence, enhanced or weakened catalytic activity, different substrate specificities, etc.

[0070] Preferably, the PCMT1 may be selected from wild-type and mutant PCMT1 with catalytic activity in bacterial, insect cell, mammalian cell, and yeast cloning expression systems. Such as PCMT1 of mammals (such as humans, mice, rats, monkeys, etc.), birds, amphibians, reptiles, plants, fungi, bacteria, and prokaryotes, wild-type PCMT1 with catalytic activity, mutant PCMT1 with catalytic activity, PCMT1 variants. The mutant PCMT1 may have characteristics such as an altered amino acid sequence, enhanced or weakened catalytic activity, different substrate specificities, etc.

[0071] Preferably, the hydrophilic amino acid is selected from any one of histidine, alanine, glycine, serine, aspartic acid, and glutamic acid; the hydrophobic amino acid is selected from any one of cysteine, phenylalanine, tryptophan, isoleucine, valine, and leucine; the amino acid with a charged group is selected from any one of lysine, arginine, aspartic acid, and glutamic acid.

[0072] The embodiments of the present invention also provide an application of the above method for quantitatively determining the activity of protein-L-isaspartate (D-aspartate)-methyltransferase (PCMT1).

[0073] Preferably, this method can quickly and efficiently screen out a large number of PCMT1 inhibitors.

[0074] In the present invention, PCMT1 transfers the methyl group on S-adenosylmethionine (SAM) to a polypeptide substrate to generate S-adenosylhomocysteine (SAH). SAH is hydrolyzed to homocysteine under the catalysis of S-adenosylhomocysteine hydrolase (SAHH), and the latter undergoes a quantitative Michael addition reaction with a fluorescent dye to generate a product with a changed fluorescence characteristic. By detecting the change in the fluorescence intensity of the product, the activity of PCMT1 is quantitatively determined. The schematic diagram of the reaction mechanism is as Figure 10 shown in B.

[0075] Example 1

[0076] A method for quantitatively determining the activity of PCMT1, and the standard working conditions of the system are as follows:

[0077] The concentration of PCMT1 is 0.6 μM;

[0078] The concentration of SAHH is 7.5 μM;

[0079] The fluorescent dye is ThioGlo4, and the concentration is 15 μM;

[0080] The polypeptide substrate is VYP-isoD-HA, and the concentration is 1 μM;

[0081] The concentration of SAM is 10 μM;

[0082] The buffer solution is 20 mM Tris-HCl, pH 7.5, 0.01% Triton X-100;

[0083] The incubation and testing temperature is 37 °C, the incubation time is 15 min, and the testing time is 20 min;

[0084] The excitation wavelength and emission wavelength are 400 nm and 465 nm;

[0085] Using the system configured as above, the method specifically includes the following steps:

[0086] (1) Preparation of buffer solution:

[0087] Prepare the buffer solution to ensure that the concentrations of each component are: 0.6 μM PCMT1, 7.5 μM SAHH, 10 μM SAM, 15 μM ThioGlo4.

[0088] The formula of the buffer solution is: 20 mM Tris-HCl, pH 7.5, adding 0.01% Triton X-100.

[0089] The prepared buffer solution needs to be thoroughly mixed before use and ensure that the concentrations of each component are accurate.

[0090] (2) Incubation step:

[0091] Take 90 μL of the prepared buffer solution and dispense it into a 384-well plate, adding it evenly to each well.

[0092] Place the 384-well plate in a 37 °C incubator and incubate it in the dark for 15 minutes.

[0093] During the incubation, ensure that the well plate is placed horizontally to ensure that the solution in each well is fully mixed.

[0094] (3) Substrate addition:

[0095] Take 10 μL of the polypeptide substrate with a concentration of 10 μM (such as VYP-isoD-HA) and carefully add it to each well using a pipette.

[0096] Use a pipette to perform up and down mixing operations to ensure that the substrate is evenly distributed in each well. The mixing operation needs to be performed 10 times to ensure that the solution is completely mixed.

[0097] (4) Fluorescence monitoring:

[0098] Place the 384-well plate in the fluorescence module of a multi-functional microplate reader, and set the excitation wavelength to 400 nm and the emission wavelength to 465 nm.

[0099] Using the fluorescence intensity mode, start the kinetic monitoring program and record the change in fluorescence intensity of each well once per minute.

[0100] The monitoring lasts for 20 minutes. During this process, fluorescence data is collected once per minute to ensure real-time reflection of the fluorescence changes during the reaction.

[0101] (5) Data analysis:

[0102] By calculating the change rate of fluorescence intensity, the fluorescence intensity growth rate of each well is obtained.

[0103] Correlate the fluorescence intensity growth rate with the enzyme activity of PCMT1 to further analyze the activity level of PCMT1 under different conditions.

[0104] Software (such as Excel or dedicated analysis software) can be used to process the fluorescence data, plot the reaction rate curve, and quantitatively analyze the activity of PCMT1.

[0105] Example 2

[0106] The schematic diagram of the solid-phase synthesis process of the polypeptide substrate VYP-isoD-HA is as Figure 3 shown, and specifically includes the following steps:

[0107] (1) Swelling of the resin: Add 30 mg of MBHA resin to 2 mL of DMF for swelling, which usually takes overnight (about 12 - 24 hours), and then remove the solvent by filtration. This step helps to ensure that the pore structure of the resin is effectively expanded, providing a good environment for subsequent reactions;

[0108] (2) Add 2 mL of 20% piperidine / DMF solution and stir for 60 minutes. Piperidine, as a deprotection reagent, can efficiently remove the Fmoc group. Then, rinse the resin 6 times with 2 mL of DMF to ensure complete removal of the Fmoc group on the resin surface and rinse off the residual solution;

[0109] (3) Amino acid condensation reaction: Add 500 μL of 1 M amino acid DMF solution, 500 μL of 1 M Oxyma DMF solution, and 77 μL of DIC (N,N'-diisopropylcarbodiimide) to the resin. Oxyma is a commonly used condensation reagent, and DIC is a common activator. The two cooperate to effectively promote the amino acid condensation reaction. React at 45 °C for 2 to 6 hours to ensure that the amino acid reacts with the amino group on the resin to form a peptide chain. After the reaction is completed, rinse the resin 6 times with 2 mL of DMF to remove unreacted reagents and by-products;

[0110] (4) Repeat steps (2), (3), and (4) until the desired polypeptide chain length is synthesized. After each synthesis, rinse the resin alternately with DMF and DCM to ensure removal of excess reagents and solvents;

[0111] (5) Isolate and purify the polypeptide: Add 2 mL of a cleavage solution of TFA / H2O (95:5) to the resin. TFA (trifluoroacetic acid) is a commonly used cleavage reagent that can break the covalent bond between the resin and the polypeptide chain, causing the polypeptide to detach from the resin. This reaction is usually stirred at room temperature for 2 hours. After the reaction is complete, remove the resin by filtration and collect the solution. Use nitrogen to remove the solvent in the solution to obtain the crude product. Dissolve the crude product in an aqueous methanol solution and adjust the pH to neutral (usually adjusted using NaOH or HCl). Finally, use preparative high-performance liquid chromatography (HPLC) for separation and purification to remove unreacted residues and impurities to obtain the purified polypeptide.

[0112] Example 3

[0113] The results of the prokaryotic expression and isolation and purification of PCMT1 are as Figure 1 shown, and specifically include the following steps:

[0114] (1) Transform the PCMT1 plasmid: Transform the PCMT1 plasmid into competent cells of the protein expression strain BL21 (Rosetta), coat it on a plate containing ampicillin, and place it in a constant temperature incubator at 37°C for culture.

[0115] (2) Primary culture: Pick a single colony and transfer it to 100 mL of LB liquid medium containing 50 μg / mL ampicillin. Culture it in a shaker at 37°C and 180 rpm for 12 hours.

[0116] (3) Scale-up culture: Take two conical flasks containing 1 L of LB liquid medium, add 1 mL of 1000× ampicillin to each of them, with a final concentration of 50 μg / mL, and mix well. Then add 50 mL of the bacterial solution cultured for 12 h to each of them, and place them in a shaker for shaking culture at 37°C and 180 rpm.

[0117] (4) Induced expression: When the exponentially growing Escherichia coli obtained by scale-up culture reaches an OD value of 0.6 - 0.8 in the bacterial solution, cool the bacterial solution in a chromatography cabinet at 4°C, and take a small amount of the bacterial solution as a sample (uninduced). Add the inducer isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 1 mM to the cooled bacterial solution, and place it in a shaker for shaking culture at 30°C and 180 rpm for 10 h to induce sufficient protein expression.

[0118] (5) Centrifugally collect the bacteria: Centrifuge the induced bacterial solution at 4°C and 4000 rpm for 10 minutes to remove the supernatant. Add 30 mL of Buffer A to resuspend the bacteria, and add PMSF at a final concentration of 1 mM and β-mercaptoethanol at 3 mM and mix well. Homogenize the resuspended bacterial solution with a high-pressure homogenizer at 4°C until the bacterial solution becomes clearer and more transparent.

[0119] (6) Centrifuge to remove cell debris: Centrifuge the disrupted bacterial solution at 14000 rpm for 30 minutes to remove cell debris. Repeat this centrifugation step three times to ensure complete removal of cell remnants.

[0120] (7) Ni-NTA affinity chromatography purification: Use an AKTA protein purification system to separate and purify the PCMT1 protein through a Ni-NTA affinity chromatography column. Pre-rinse the affinity column with 50 mL of Buffer B, and then equilibrate it with Buffer A. Use a sample loading pump to load the disrupted solution, and elute the miscellaneous proteins through the column until the UV absorption value at A280 nm returns to zero to ensure no impurities. Use Buffer B to elute the target protein. (Buffer A: 25 mM HEPES, 500 mM NaCl, 500 mM TCEP, 20 mM imidazole, pH 8.0; Buffer B: 25 mM HEPES, 500 mM NaCl, 500 mM TCEP, 400 mM imidazole, pH 8.0;)

[0121] (8) Dialyze to remove imidazole: Transfer the eluted target protein into a dialysis bag and place it in 100 times the volume of dialysis solution (HEPES pH 8.0, concentration 25 mM; NaCl concentration 500 mM). Dialyze at 4°C for 4 hours to remove imidazole and other small molecule impurities.

[0122] (9) Remove the His tag: Add thrombin to the dialyzed PCMT1 protein according to the ratio of PCMT1:thrombin = 1000:1. Place the protein solution in 100 times the volume of dialysis solution of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and perform an enzymatic digestion reaction at room temperature for 12 hours. Remove the His tag of the PCMT1 protein by thrombin to obtain the final purified protein.

[0123] (10) Purity verification: Use 12% SDS-PAGE for gel electrophoresis verification.

[0124] Example 4

[0125] The prokaryotic expression and separation and purification results of SAHH are as Figure 2 shown, specifically including the following steps:

[0126] (1) Transformation of SAHH plasmid: The SAHH plasmid was transformed into competent cells of protein expression strain BL21 (Rosetta), spread on a plate containing ampicillin, and placed in an incubator at 37°C for cultivation.

[0127] (2) Primary cultivation: A single colony was picked and placed in 100 mL of LB liquid medium containing 50 μg / mL ampicillin, and cultured in a shaker at 37°C and 180 rpm for 12 h.

[0128] (3) Scale-up cultivation: Two conical flasks containing 1 L of LB liquid medium were taken, and 1 mL of 1000× ampicillin was added to each of them, with a final concentration of 50 μg / mL, and mixed well. Then, 50 mL of the bacterial solution cultured for 12 h was added to each of them, and cultured in a shaker at 37°C and 180 rpm.

[0129] (4) Induced expression: When the Escherichia coli in the scale-up cultivation reached the logarithmic growth phase, that is, when the OD value of the bacterial solution was 0.6 - 0.8, the bacterial solution was placed in a chromatography cabinet at 4°C for cooling, and a small amount of the bacterial solution was taken as a sample (not induced). Isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.4 mM was added to the cooled bacterial solution, and cultured in a shaker at 30°C and 180 rpm for 10 h to induce sufficient protein expression.

[0130] (5) Centrifugation to collect bacteria: The bacterial solution was centrifuged at 4°C and 4000 rpm for 10 min. After centrifugation, the supernatant was discarded, 30 mL of Buffer A was added to resuspend the bacteria, and 1 mM PMSF and 3 mM β-mercaptoethanol were added and mixed well. The resuspended bacterial solution was broken using a high-pressure homogenizer pre-cooled to 4°C, and the broken bacterial solution was clearer and more transparent than before breaking.

[0131] (6) Centrifugation to remove cell debris: The broken bacterial solution was centrifuged at 14000 rpm for 30 min, and this was repeated three times.

[0132] (7) Ni-NTA affinity chromatography purification: Using an AKTA protein purification system, the SAHH protein was separated and purified through a Ni-NTA affinity chromatography column. The Ni-NTA was pre-equilibrated. After flushing the column with 50 mL of Buffer B, the column was continuously flushed with Buffer A for equilibration. A sample loading pump was used for sample loading, and this was repeated twice. Subsequently, Buffer A was used to flush out the contaminating proteins until no substances appeared in the eluate (monitored by ultraviolet absorption at A280 nm), and then Buffer B was used to flush out the target protein. (Buffer A: 25 mM HEPES, 500 mM NaCl, 500 mM TCEP, 20 mM imidazole, pH 8.0; Buffer B: 25 mM HEPES, 500 mM NaCl, 500 mM TCEP, 400 mM imidazole, pH 8.0;)

[0134] (8) Dialysis to remove imidazole: The target protein was loaded into a dialysis bag and placed in 100-fold volume of dialysis buffer (the final concentration of NaH 2 PO 4 / Na 2 HPO 4 with a final concentration of 50 mM at pH 7.0 and the final concentration of NaCl of 500 mM), and dialyzed at 4 °C for 4 h in a chromatography cabinet to remove the imidazole in the protein.

[0135] (9) Removal of the His tag: Thrombin was added to the dialyzed PCMT1 protein at a ratio of PCMT1:thrombin = 50:1 and placed in 100-fold volume of dialysis buffer (20 mM KH 2 PO 4 / K 2 HPO 4 at pH 7.2, 100 mM NaCl), and digested at 4 °C for 2 h, and then digested in the same dialysis buffer at 4 °C for 12 h.

[0136] (10) Purity verification: Verified using 12% SDS-PAGE.

[0137] Example 5

[0138] Determine the types of buffer solutions used, ionic concentrations, pH, and types and concentrations of surfactants, specifically including the following steps:

[0139] (1) Prepare different types of buffer solutions

[0140] Prepare buffer solutions with the following specific components: 1 μM PCMT1; 7.5 μM SAHH; 100 μM SAM; 15 μM ThioGlo4 (fluorescent dye)

[0141] Buffer solution formulation: A: 20 mM Tris-HCl, pH 7.5, 0.01% Triton X-100

[0142] B: 20 mM Tris-HCl, pH 7.5 (without surfactant)

[0143] (2) Incubate the samples

[0144] Take 90 μL of the prepared buffer solution and add it to a 384-well plate. Incubate in the dark at 37 °C for 15 minutes.

[0145] (3) Add saturated concentration of VYP-iso-DHA (peptide substrate) to initiate the reaction

[0146] Prepare a 100 μM solution of VYP-iso-DHA (peptide substrate). Take 10 μL of the peptide substrate solution and add it to each well in the 384-well plate. Mix the solution up and down 10 times using a pipette to ensure uniformity.

[0147] (4) Fluorescence intensity measurement

[0148] On a multi-functional microplate reader, select the Fluorescence intensity module. Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm. Perform a kinetic cycle and monitor the fluorescence intensity once per minute for 20 minutes.

[0149] (5) Data analysis

[0150] Evaluate the effects of the two buffer solutions on enzyme activity based on the fluorescence intensity growth rate of each buffer solution used. Select the buffer solution with the highest fluorescence intensity growth rate and determine it as the optimal condition.

[0151] Example 6

[0152] Under saturated substrate concentration, test the effect of temperature on the reaction curve, including the following steps:

[0153] (1) Prepare the buffer solution

[0154] Prepare the buffer solution with the following specific components: 1 μM PCMT1; 7.5 μM SAHH; 100 μM SAM; 15 μM ThioGlo4 (fluorescent dye)

[0155] Buffer solution formulation: 20 mM Tris-HCl, pH 7.5, 0.01% Triton X-100

[0156] (2) Incubate the samples

[0157] Take 90 μL of the prepared buffer solution and add it to a 384-well plate.

[0158] Incubate in the dark for 15 minutes at 26 °C and 37 °C respectively.

[0159] (3) Initiate the reaction

[0160] Prepare a 100 μM solution of VYP-iso-DHA (peptide substrate).

[0161] Take 10 μL of the peptide substrate solution and add it to the pre-incubated 384-well plate respectively.

[0162] Use a pipette to mix the solution up and down 10 times to ensure the solution is uniform.

[0163] (4) Fluorescence intensity measurement

[0164] Use a multimode microplate reader to select the Fluorescence intensity module.

[0165] Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm.

[0166] Establish a kinetic cycle and monitor the fluorescence intensity once per minute for 20 minutes.

[0167] (5) Data analysis

[0168] Draw a reaction curve based on the fluorescence intensity growth rate at different temperatures.

[0169] Compare the curve slopes at 26 °C and 37 °C and analyze the effect of temperature on the enzymatic reaction activity of PCMT1.

[0170] Example 7

[0171] Test the reaction linear range of PCMT1 concentration at saturated substrate concentration and determine the PCMT1 usage concentration, including the following steps:

[0172] (1) Prepare the buffer solution system

[0173] Prepare the following buffer solutions (each group corresponds to a different concentration of PCMT1):

[0174] PCMT1 concentration: 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM

[0175] 7.5 μM SAHH

[0176] 100 μM SAM (saturated substrate concentration)

[0177] 15 μM ThioGlo4 (fluorescent dye)

[0178] Buffer formulation: 20 mM Tris-HCl, pH 7.5, 0.01% Triton X-100

[0179] (2) Incubate the samples

[0180] Take 90 μL of the prepared buffer solution and add it to a 384-well plate.

[0181] Incubate in the dark at 37 °C for 15 minutes.

[0182] (3) Initiate the reaction

[0183] Prepare a 100 μM VYP-iso-DHA (peptide substrate) solution.

[0184] Take 10 μL of the peptide substrate solution and add it to the corresponding 384-well plate for each group.

[0185] Use a pipette to mix the solution up and down 10 times to ensure uniformity.

[0186] (4) Fluorescence intensity measurement

[0187] Use a multifunctional microplate reader to select the Fluorescence intensity module.

[0188] Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm.

[0189] Establish a kinetic cycle and monitor the fluorescence intensity once per minute for 20 minutes.

[0190] (5) Data analysis

[0191] Calculate the reaction rate: Determine the enzymatic reaction rate at each PCMT1 concentration based on the growth rate of the fluorescence intensity.

[0192] Plot the reaction linear range graph: Plot the PCMT1 concentration against the corresponding reaction rate as a curve to observe its linear range.

[0193] Determine the working concentration: Select a suitable PCMT1 concentration as the standard concentration for subsequent experiments based on the linear range and experimental requirements.

[0194] Example 8

[0195] Under saturated substrate concentration, test the effects of DMSO and ThioGlo4 concentrations on the reaction curve, including the following steps:

[0196] (1) Prepare the buffer solution system

[0197] Prepare the following buffer solutions:

[0198] PCMT1 concentration: 0.6 μM

[0199] SAHH concentration: 7.5 μM

[0200] SAM concentration: 100 μM (saturated substrate concentration)

[0201] Buffer formulation: 20 mM Tris-HCl, pH 7.5, 0.01% Triton X-100

[0202] (2) Design experimental conditions

[0203] DMSO concentration groups: Prepare buffer solutions containing 1%, 2%, 4%, 6%, and 8% DMSO.

[0204] ThioGlo4 concentration groups: Prepare buffer solutions with ThioGlo4 concentrations of 10 μM, 15 μM, 20 μM, and 25 μM respectively.

[0205] Repeat the experiment three times for each combination to ensure data reliability.

[0206] (3) Incubate the samples

[0207] Take 90 μL of the buffer solution prepared under the corresponding conditions and add it to a 384-well plate.

[0208] Incubate in the dark at 37 °C for 15 minutes.

[0209] (4) Initiate the reaction

[0210] Prepare a 100 μM VYP-iso-DHA (peptide substrate) solution.

[0211] Take 10 μL of the peptide substrate solution and add it to each corresponding group in the 384-well plate.

[0212] Use a pipette to mix the solution up and down 10 times to ensure uniform solution.

[0213] (5) Fluorescence intensity measurement

[0214] Use a multi-functional microplate reader to select the Fluorescence intensity module.

[0215] Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm.

[0216] Establish a kinetic cycle and monitor the fluorescence intensity once per minute for 20 minutes.

[0217] (6) Data analysis

[0218] Reaction rate calculation: Calculate the reaction rate under each group of conditions based on the growth rate of the fluorescence intensity.

[0219] Data comparison: Analyze the effects of different DMSO concentrations and ThioGlo4 concentrations on the reaction rate respectively.

[0220] Determine the optimal conditions:

[0221] For DMSO, determine the optimal concentration range that does not affect enzyme activity and is compatible with the system.

[0222] For ThioGlo4, select the optimal concentration with the strongest fluorescence signal and the least background interference.

[0223] Example 9

[0224] Determine the K m value of the substrate methyl donor SAM, which specifically includes the following steps:

[0225] Fix the polypeptide substrate at a saturated concentration (10 μM), and vary the concentration of SAM in the range of 0 - 100 μM to measure the K m .

[0226] (1) Prepare the buffer solution

[0227] Prepare the buffer solution with the following specific components: 0.6 μM PCMT1; 7.5 μM SAHH; 10 μM VYP-iso-DHA (polypeptide substrate); 15 μM ThioGlo4 (fluorescent dye)

[0228] Buffer solution formula: 20 mM Tris-HCl, pH 7.5; 0.01% Triton X-100

[0229] (2) Incubate the samples

[0230] Take 90 μL of the prepared buffer solution and add it to a 384-well plate. Incubate in the dark at 37 °C for 15 minutes.

[0231] (3) Dilute the SAM solution

[0232] Start diluting SAM from a concentration of 1000 μM with a 2-fold gradient to obtain a series of concentrations (0 - 100 μM).

[0233] (4) Add different concentrations of SAM to initiate the reaction

[0234] Take 10 μL of SAM solutions with different concentrations and add them to each well in the 384-well plate respectively. Use a pipette to mix the solution up and down 10 times to ensure the solution is uniform.

[0235] (5) Measure the fluorescence intensity

[0236] On a multifunctional microplate reader, select the Fluorescence intensity module. Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm. Conduct a kinetic cycle, monitoring the fluorescence intensity once per minute for 20 minutes continuously.

[0237] (6) Data analysis and K m value calculation

[0238] Calculate the enzyme activity of PCMT1 according to the fluorescence intensity growth rate of each well. Fit the data based on the enzyme kinetics curve to obtain the K m value.

[0239] Example 10

[0240] Determine the K m value of the polypeptide substrate VYP-isoD-HA, which specifically includes the following steps:

[0241] At a fixed substrate SAM at a saturated concentration (100 μM), the concentration of the polypeptide substrate VYP-iso-DHA varies within the range of 0 - 100 μM to determine the K m of the polypeptide substrate VYP-isoD-HA.

[0242] (1) Prepare the buffer solution

[0243] Prepare the buffer solution with the following specific components: 0.6 μM PCMT1; 7.5 μM SAHH; 10 μM SAM (methyl donor); 15 μM ThioGlo4 (fluorescent dye)

[0244] Buffer solution formula: 20 mM Tris-HCl, pH 7.5; 0.01% Triton X-100

[0245] (2) Incubate the samples

[0246] Take 90 μL of the prepared buffer solution and add it to a 384-well plate. Incubate in the dark at 37 °C for 15 minutes.

[0247] (3) Dilute the polypeptide solution

[0248] Start diluting the polypeptide from a concentration of 100 μM with a 2-fold gradient to obtain a series of concentrations (0 - 100 μM).

[0249] (4) Add polypeptides at different concentrations to initiate the reaction

[0250] Take 10 μL of polypeptide solutions at different concentrations and add them to each well in the 384-well plate respectively. Use a pipette to mix the solution up and down 10 times to ensure the solution is uniform.

[0251] (5) Fluorescence intensity measurement

[0252] On a multifunctional microplate reader, select the Fluorescence intensity module. Set the excitation wavelength to 400 nm and the emission wavelength to 465 nm. Perform a kinetic cycle, monitoring the fluorescence intensity once per minute for 20 minutes continuously.

[0253] (6) Data analysis and K m value calculation

[0254] Calculate the enzyme activity of PCMT1 according to the fluorescence intensity growth rate of each well. Fit the data based on the enzyme kinetic curve to obtain the K m value.

[0255] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for quantitatively determining the activity of protein-L-isoaspartate (D-aspartate)-methyltransferase (PCMT1), the method being for non-disease diagnosis and treatment purposes, characterized in that: The method involves transferring the methyl group on S-adenosylmethionine (SAM) to a polypeptide substrate via PCMT1 to generate S-adenosylhomocysteine ​​(SAH), which is then hydrolyzed to homocysteine ​​under the catalysis of S-adenosylhomocysteine ​​hydrolase (SAHH), and then undergoes a quantitative Michael addition reaction with a fluorescent dye. The activity of PCMT1 is quantitatively determined by detecting changes in the product fluorescence signal. The general structural formula of the polypeptide substrate is Among them, R1 and R2 are selected from at least one of hydrophilic amino acids, hydrophobic amino acids, amino acids with charged groups, methylated or phosphorylated amino acids, fatty acid-containing amino acids, and cyanated amino acids.

2. The method according to claim 1, characterized in that: The steps include: S1. In a Tris-HCl buffer system with a pH value of 5.5 to 10.5, SAM and peptide substrates were introduced for catalysis by PCMT1, and the enzyme catalytic reaction was incubated at a temperature of 15 to 50°C for 1 to 120 minutes to generate SAH; the concentration range of peptide substrate and SAM was 0.1 to 10K m ; S2. adding a hydrolase to SAH to hydrolyze it to generate homocysteine; the concentration range of the hydrolase is 0.1 to 500 μM; S3. adding homocysteine ​​to a fluorescent dye to react and generate a fluorescent product; the concentration of the fluorescent dye ranges from 0.1 to 500 μM; S4. The activity of PCMT1 can be quantitatively determined by detecting changes in fluorescence signals.

3. The method according to claim 2, characterized in that The general structural formula of the fluorescent dye is: Among them, R1 is hydroxyl, straight-chain or branched alkyl having 1 to 10 carbon atoms, aryl, halogenated alkyl or heterocyclic group containing oxygen, nitrogen or sulfur; R5 is ester, amide, cyano, sulfonamide, sulfonate or its derivative; R2, R3 and R4 are selected from hydrogen, alkyl, aryl, halogen, hydroxyl, methoxy, nitro and amino.

4. The method according to claim 3, characterized in that The polypeptide substrate is selected from at least one of KQVV-isoD-SAYEVIK, A-isoD-QLTEEQIAEFK, TS-isoD-TSKY, VYP-isoD-HA, VYP-isoD-DA, KASA-isoD-LAKY, VYP-isoD-AA, VYP-isoD-CA, VYP-isoD-SA, and VYP-isoD-RR.

5. The method according to claim 4, characterized in that The system further comprises at least one of a surfactant and an anti-interference substance.

6. The method according to claim 5, characterized in that The S-adenosylhomocysteine ​​hydrolase (SAHH) can be selected from wild-type and mutant SAHHs from any species and having catalytic activity.

7. The method according to claim 6, characterized in that The PCMT1 can be selected from wild-type and mutant PCMT1 with catalytic activity in bacteria, insect cells, mammalian cells and yeast cloning expression systems.

8. The method according to claim 1, characterized in that The hydrophilic amino acid is selected from any one of histidine, alanine, glycine, serine, aspartic acid, and glutamic acid; the hydrophobic amino acid is selected from any one of cysteine, phenylalanine, tryptophan, isoleucine, valine, and leucine; and the amino acid with a charged group is selected from any one of lysine, arginine, aspartic acid, and glutamic acid.

9. Use of the method for quantitatively determining the activity of protein-L-isoaspartate (D-aspartate)-methyltransferase (PCMT1) according to any one of claims 1 to 8.

10. The use according to claim 9, characterized in that: This method can quickly and efficiently screen a large number of PCMT1 inhibitors.

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