Transglutaminase: Mechanistic features of the active site as determined by kinetic and inhibitor studies

1. The Km values for the transglutaminase-catalyzed (amine: R-glutamine transferase (hydrolyzing) hydrolysis and hydroxylamine incorporation reactions and the Kn values for inhibition by carbobenzoxy-(CBZ)-amino acids and CBZ-peptides, all obtained as functions of pH, reveal an enzyme group of pK 7.5 to 8.5. Inhibitor specificity studies indicate that this group is involved in binding substrate at the peptide bond formed through the α-amino group of the glutamine residue. 2. Inhibitor constants for chloroacetyl amino acids show no variation with pH. It is suggested that these inhibitors, as well as chloroacetic acid, are bound to an enzyme active center group through the carbonyl carbon of their chloroacetyl position. The change in Ki values for chloroacetic acid at about pH 6.5 has been attributed to an induced shift in pK of another group which is in the close structural vicinity. 3. This group, tentatively identified as an-SH on the basis of its sensitivity to thiol reagents, has an apparent pK of around 6.5, as judged from activity titration versus p H with chloroacetamide, decrease in vmax. with pH and induced shift in its pK with chloroacetic acid. 4. The efficient protection against thiol reagent inhibition by substrate supports a supposition that this -SH group forms a covalent bond with substrate during the catalytic reaction.