THE ROLE OF METHIONINE IN ALPHA-CHYMOTRYPSIN-CATALYSED REACTIONS.
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.
D000215:Acylation; D000577:Amides; D000596:Amino Acids; D001669:Biochemical Phenomena; D001671:Biochemistry; D002384:Catalysis; D002918:Chymotrypsin; D007093:Imidazoles; D007700:Kinetics; D008715:Methionine; D009596:Nitrophenols; D010504:Periodic Acid; D012106:Research; D013053:Spectrophotometry; D014364:Tryptophan / Q000031:analogs & derivatives; D014443:Tyrosine / Q000031:analogs & derivatives; D014461:Ultracentrifugation; D014633:Valine
D000577:Amides; D000596:Amino Acids; D007093:Imidazoles; D009596:Nitrophenols; D010504:Periodic Acid; C016952:N-acetyltryptophanamide; D014443:Tyrosine; C025166:tryptophan ethyl ester; C008642:4-nitrophenyl acetate; D014364:Tryptophan; C051772:ethyl tyrosine ester; D008715:Methionine; C009288:metaperiodate; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D014633:Valine
AMIDES; AMINO ACIDS; BIOCHEMISTRY; CATALYSIS; CHYMOTRYPSIN; EXPERIMENTAL LAB STUDY; IMIDAZOLES; METHIONINE; PERIODIC ACIDS; SPECTROPHOTOMETRY; TRYPTOPHAN; ULTRACENTRIFUGATION; VALINE