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 normal 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,,/K,,, value for the thioxo peptide is 1100-fold lower than for the corresponding 0x0 peptide. This difference is entirely due to k,,,. Because the rotation about the thioxo amide bond is about 12.5 kJ mol-' 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 4-nitroanilide and dipeptidyl peptidase IV 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,-PI 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 0x0 and the thioxo form.Modification of the backbone of biologically active peptides has become increasingly important in the design of analogues possessing greater potency and enzymatic stability. Peptide bond modifications often induce drastic changes in flexibility of the peptide backbone. In contrast X-ray [l], infrared [2], CD [3] and NMR [4J studies of simple thioxo di-and tri-peptides have revealed that a thioxo amide mainly adopts a Z planar configuration similar to an amide, except that the thioxo-carbonyl bond is longer (C=O = 0.124 nm, C=S = 0.164 nm) [l, 51. Furthermore, the larger covalent [6] and van der Waals [7] radius of sulfur restricts the allowable $,iy angles in the vicinity of thioxo amides [8]. Thioxo peptides are also known to display a higher barrier of rotation about the C-N bond by 8-12 kJ/mol [9]. Amides and thioxo amides differ also in their hydrogen-bond forming properties. Thioxo amides are stronger acids [lo] but weaker bases than amides [ll]. Nevertheless, the introduction of a thioxo amide bond is a nearly isosteric substitution for an amide bond [12] often accompanied with surprising effects on the conformaCorrespondence to M. Schutkowski, Max-Planck-Gesellschaft zu Forderung der Wissenschaft e. V.,