Enzyme assay serine proteases

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

For many enzymes of clinical or commercial importance, chromogenic substrates or cofactors have been developed to allow enzyme assays to be analysed photometrically. For example the compounds tosyl-glycyl-prolyl-lysyl-4-nitroacetanilidoacetate, or tosyl-glycyl-arginyl-4-nitroacetanilide can be used to assay the dinically important serine proteases plasmin and thrombin. 

Other enzymes are also useful indices of liver pathology. Serum alkaline phosphatase is often a useful indicator of liver and bone disease. The alkaline phosphatases are a diverse group of enzymes that catalyze reactions in which a phosphate is removed from a phosphate ester, especially at an alkaline pH. Physicians don t care about this. They do care that serum alkaline phosphatase levels often rise with bone breakdown (as in tumor infiltration) and in liver disease, especially where tliere is obstruction of the bile duct. Acid phosphatase is particularly rich in the prostate. A rise in its serum levels provides a test as to the presence of prostate carcinoma. This test has largely been replaced by assay for Prostate Specific Antigen (PSA), a serine protease that is elevated in prostatic carcinoma. 

In the second example, two primary, in vitro serine protease assays with identical /Miitroaniline-based chromogenic (A405) readouts were either assayed separately or multiplexed. Positive control inhibitors specific to each enzyme were added to the assay at an appropriate concentration to determine assay... 

Acetylcholinesterase is mechanistically related to serine proteases and involves acylation of Ser and contains the catalytic triad Ser -His -Glu. Precise data for acetylation by the natural substrate acetylcholine is difficult to obtain due to lack of a convenient assay method, so the mechanistically equivalent surrogate acetylthio-choline has frequently been used to probe mechanistic aspects of this enzyme [15]. To explore proton transfer reactions that accompany acetylation by this substrate, solvent isotope effect measurements and proton inventories on k /Km have been conducted [16, 17]. The isotope effects are near unity and the proton inventories bowed-upwards, suggesting that the transition state for kjKm is a virtual transi-... 

PSA is one of the most widely used cancer biomarkers. It is a chymotryp-sin-like serine protease that is produced by epithelial cells of the prostate gland and secreted into the prostatic fluid. Prostate-cancer invasion disrupts the epithelial membrane barrier leading to elevated serum levels of PSA. Detection of PSA in blood can therefore be useful in the diagnosis of prostate abnormalities and for evaluation of prostate cancer therapy efficacy [21]. Two different forms of PSA are immunologically detectable the free form (MW 34 kDa) and a complex with a-l-antichymotrypsin (MW 96 kDa). Diagnostic assays developed for detection of PSA (e.g., enzyme-linked immunosorbent assays) detect total PSA concentrations down to 0.1 ngmL [22,23]. 

AT,r,=41 0.7 IIM. These rates were also pH dependent, with Ka = 6.58, in reasonable agreement with the catalytic Ka value for a serine protease. The actual inactivation rate was determined from rescue experiments. At various times t following addition of suicide substrate inhibitor to enzyme, 10 vaM of the nucleophile )6-mercaptoethanol was added. This nucleophile reacted rapidly with excess ynenol lactone, allowing any enzyme not inactivated to deacylate to regenerate active enzyme, as shown in Fig. 13.2. The inactivation rates were also saturable, giving 4 or /Tinact = 0.0037 0.0001 s and inact = 0.63 0.08 (xM. Gel filtration of the enzyme-inhibitor mixture before full inactivation could occur, followed by dilution into assay conditions, allowed determination of the deacylation rate, 3 = 0.0056 s The pH dependence of this rate was also determined and found to have a Ka value of 7.36. This value was in excellent agreement with the catalytic p a value, providing further evidence for the role of enzyme catalysis in the mechanism of inactivation. 

That the inactivation was active-site directed was also established in several ways. As mentioned above, the pA a values of k2 and k, were consistent with the pKa value of catalytic activity for a serine protease. Difference spectra of enzyme with inhibitor showed the reactive product being formed in the presence of enzyme. Rates of inhibition decreased in the presence of a known competitive inhibitor, elastatinal (Okura et al., 1975). The reactive intermediate was generated by mild alkaline hydrolysis and added to assay buffer at a concentration 25 times higher than the Ki of the ynenol lactone. Enzyme and substrate were added to the mixture, and neither inhibition nor time-dependent inactivation was observed. Therefore, inactivation was unlikely to occur by enzymatic release of the reactive intermediate followed by nonspecific alkylation outside the active site. 

The serine protease from Thermus caldophilus strain GK-24 [292] gave maximum activity at 90 °C in 20 min assays and had a broad pH optimum with casein as substrate. The enzyme showed hydrolytic activity on some small peptide substrates (e.g. CBZ-L-leu-L-tyr-NHj) and also possessed esterase activity. Hydrolysis of synthetic chromogenic peptides and esters is also a property of the recently described serine protease, caldolase, from Thermus strain ToK3 [293]. This enzyme contained 10% carbohydrate and four disulphide bonds, but neither calcium nor zinc were detected in the purified enzyme. Thermostability of the enzyme was high in 0.4 M NaCl, but in low ionic strength buffer rapid thermal denaturation occurred at 75 °C. Work has also been undertaken in this laboratory... 

Pardee and co-workers (225,225a,227,227a) have investigated the activity of 9 bacterial enzymes after infection of E. coli with various T phages, i.e., apyrase, ribonuclease, deoxyribonuclease, alkaline and acid proteases, pyruvic oxidase, formic dehydrogenase, serine deaminase, and catalase. The difficulties of assay of labile enzymes in the broken cell systems are indicated by the early report of an increase in apyrase activity (225) which later was attributed to a technical defect in the... 

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