INFLUENCE ON PROLINE-SPECIFIC ENZYMES OF A SUBSTRATE CONTAINING THE THIOXOAMINOACYL-PROLYL PEPTIDE-BOND

Dipeptidyl peptidase IV from porcine kidney and aminopeptidase P from Escherichia coli can utilize thioxoalanyl-proline 4-nitroanilide but with decreased kinetic constants compared to the nor mal substrates. Product analysis showed that exclusively thioxoalanyl-proline was liberated in the case of dipeptidyl peptidase IV catalysis and thioxo-alanine in the case of aminopeptidase-P-mediated thioxo peptide bond hydrolysis. For the proline-specific aminopeptidase P the k(cat)/K-m value for the thioxo peptide is 1100-fold lower than for the corresponding oxo peptide. This difference is entirely due to k(cat). Because the rotation about the thioxo amide bond is about 12.5 kJ mol(-1) more difficult than rotation about an amide bond, these data support a mechanism involving rate-limiting rotation about the scissile peptide bond. It was found that the specificity rate constant for the reaction of thioxoalanyl-proline LF-nitroanilide and dipeptidyl peptidase TV is 100-1000-fold lower compared to the corresponding rate constant for alanyl-proline 4-nitroanilide. This remarkable effect is interpreted in terms of a distorted binding of the transition state for the thioxo substrate. The hydrolysis of the thioxo substrate by dipeptidyl peptidase IV is isomer-specific. The conformation about the nonscissile P-2-P-1 thioxo amide bond has to be in trans for successful cleavage of the scissile peptide bond. We can now directly compare the rotational energy barrier of the prolyl peptide bond for the oxo and the thioxo form.