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Kinetics and mechanism of catalysis by proteolytic enzymes. The kinetics of hydrolysis of esters of gamma-guanidino-L-alpha-toluene-p-sulphonamidobutyric acid by bovine trypsin and thrombin.

ABSTRACT: 1. Esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid (alpha-N-toluene-p-sulphonyl-l-norarginine) have been synthesized and shown to be hydrolysed by bovine trypsin and thrombin. As substrates for these enzymes, they were better than esters of alpha-N-toluene-p-sulphonyl-l-homoarginine or of alpha-N-toluene-p-sulphonyl-l-ornithine but not as good as esters of alpha-N-toluene-p-sulphonyl-l-arginine. 2. With trypsin as catalyst, the methyl and propyl esters are hydrolysed at the same rate at high substrate concentrations and hence deacylation of the acyl-enzyme appears to be rate-determining. In the presence of thrombin, however, the methyl ester is hydrolysed much faster than the n-propyl ester. 3. The variation of k(0) with pH indicates that groups with pK((app.)) values of 7.05+/-0.02 and 6.53+/-0.02 must be dissociated in trypsin and thrombin respectively for hydrolysis to proceed. 4. Activation constants have been determined for the trypsin-catalysed hydrolysis of methyl gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyrate and have been compared with the corresponding constants for the hydrolysis of homologous substrates. 5. Cholate increases k(0) and decreases K(m); the effects are more pronounced with thrombin than with trypsin.

SUBMITTER: Baird JB

PROVIDER: S-EPMC1207211 | biostudies-other | 1965 Sep

REPOSITORIES: biostudies-other

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Project description:Nucleoside triphosphate (NTP) hydrolysis is a key reaction in biology. It involves breaking two very stable bonds (one P-O bond and one O-H bond of water), in either a concurrent or a sequential way. Here, we systematically examine how protonation of the triphosphate affects the mechanism of hydrolysis.The hydrolysis reaction of methyl triphosphate in vacuum is computed with protons in various numbers and position on the three phosphate groups. Protonation is seen to have a strong catalytic effect, with the reaction mechanism depending highly on the protonation pattern.This dependence is apparently complicated, but is shown to obey a well-defined set of rules: Protonation of the ?- and ?-phosphate groups favors a sequential hydrolysis mechanism, whereas ?-protonation favors a concurrent mechanism, the two effects competing with each other in cases of simultaneous protonation. The rate-limiting step is always the breakup of the water molecule while it attacks the ?-phosphorus, and its barrier is lowered by ?-protonation. This step has significantly lower barriers in the sequential reactions, because the dissociated ?-metaphosphate intermediate (P?O3-) is a much better target for water attack than the un-dissociated ?-phosphate (-P?O42-). The simple chemical logic behind these rules helps to better understand the catalytic strategy used by NTPase enzymes, as illustrated here for the catalytic pocket of myosin. A set of rules was determined that describes how protonating the phosphate groups affects the hydrolysis mechanism of methyl triphosphate: Protonation of the ?- and/or ?- phosphate groups promotes a sequential mechanism in which P-O bond breaking precedes the breakup of the attacking water, whereas protonation of the ?-phosphate promotes a concurrent mechanism and lowers the rate-limiting barrier of water breakup. The role played by individual protein residues in the catalytic pocket of triphosphate hydrolysing enzymes can be assigned accordingly.

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Kinetics and mechanism of catalysis by proteolytic enzymes. The kinetics of hydrolysis of esters of gamma-guanidino-L-alpha-toluene-p-sulphonamidobutyric acid by bovine trypsin and thrombin.

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