alpha-chymotrypsin and tryptophan-ethyl-ester

The hydrolysis of L-tryptophane ethyl ester catalyzed by alpha-chymotrypsin and the effect of ethyl ster of D-tryptophane on the course of the reaction were studied. A kinetic pattern of a three-step enzymatic reaction based on the assumption that the enzyme complex with the protonated form of the substrate is the only reactive one, was proposed. It was shown that the limiting step of the enzymatic reaction consists in a formation of intermediate acyl enzyme. The pH-dependence of the bimolecular rate constant (kcat/Km) for the enzymatic hydrolysis of L-tryptophane ethyl ester is bell-shaped and is described by a pattern including ionization of two groups with pKa values of 7,0 +/- 0,1; and 7,5 +/- 0,1; the value of pKa equal to 7,5 +/- 0,1 corresponds to substrate ionization (pKas for the amino group of L-tryptophane ethyl ester is 7,6). The constants for the binding of protonated and non-protonated substrate forms by the enzyme were calculated from the step-wise dependence of the Km values of pH. An analysis of the bell-shaped dependence of the catalytic constant of enzymatic hydrolysis included determination of pK values of the ionogenic groups of the enzyme--substrate complex (pK'a = 6,8 +/- 0,1 and pK''a = 7,3 +/- 0,1).

Topics: Chymotrypsin; Hydrogen-Ion Concentration; Kinetics; Mathematics; Substrate Specificity; Tryptophan

1. The reaction of alpha-chymotrypsin with sodium periodate at pH5.0 has been investigated. The enzyme consumes 2 moles of periodate/mole, and there is a concomitant fall in enzymic activity (with respect to l-tyrosine ethyl ester) to 55% of that of the native enzyme. After 3hr. no further change is observed in periodate uptake or in catalytic activity. 2. The oxidized enzyme is a homogeneous preparation of partially active chymotrypsin. 3. In the oxidized enzyme, one of the two methionine residues in the molecule has been converted into its sulphoxide. It is this reaction only that is responsible for the loss of activity. 4. The rate constants for the enzyme-catalysed acylation and deacylation reactions are unaltered by oxidation of the enzyme, both for a non-specific substrate (p-nitrophenyl acetate), and for three specific substrates: N-acetyl-l-tryptophan ethyl ester, N-acetyl-l-tryptophanamide and N-acetyl-l-valine ethyl ester. 5. The K(m) values for the aromatic substrates with the oxidized enzyme are twice those with the native enzyme. No change in Michaelis constant is seen for the non-aromatic substrate N-acetyl-l-valine ethyl ester. 6. The evidence points to the oxidized methionine residue in the modified enzyme being situated in the locus of the active site at which aromatic (or bulky) side chains of the substrates are bound.

Topics: Acylation; Amides; Amino Acids; Biochemical Phenomena; Biochemistry; Catalysis; Chymotrypsin; Imidazoles; Kinetics; Methionine; Nitrophenols; Periodic Acid; Research; Spectrophotometry; Tryptophan; Tyrosine; Ultracentrifugation; Valine

Topics: Chemical Phenomena; Chemistry; Chymotrypsin; Enzymes; Hydrolysis; Kinetics; Research; Tryptophan

Mast cells contain an enzyme which hydrolyzes 3-chloroacetoxy-2-naphthoic acid anilide. By using highly purified mast cells isolated by differential centrifugation in high density sucrose solutions we have been able to study this enzymatic activity in more detail. The enzyme has properties similar to those of chymotrypsin: Chymotrypsin will hydrolyze the histochemical substrate, and the chymotrypsin and mast cell activities with this substrate are similarly inhibited by diisopropylfluorophosphate. The mast cell enzyme is capable of hydrolyzing the N-acetyl esters of tryptophan, tyrosine, and phenylalanine, the relative rates of hydrolysis being similar to those seen with chymotrypsin. A characteristic trypsin substrate, p-toluenesulfonyl arginine methyl ester, is not acted upon by the mast cell enzyme or chymotrypsin. The pH activity curve of the new cell enzyme is similar to that of chymotrypsin as determined with N-acetyl-L-tryptophan ethyl ester as substrate.

Topics: Chymotrypsin; Mast Cells; Naphthalenes; Trypsin; Tryptophan; Tyrosine