Effect of electron withdrawing substituents on substrate hydrolysis by and inhibition of rat neutral endopeptidase 24.11 (enkephalinase) and thermolysin

A series of N-acylphenylalanylglycine dipeptides were synthesized and examined as substrates for neutral endopeptidase 24.11 (NEP) and thermolysin. Those N-acyl dipeptides containing an N-acyl group derived from an acid whose pK_a is below 3.5 were considerably more reactive with both enzymes than those peptides containing an N-acyl group derived from an acid whose pK_a is above 4. The data are interpreted to suggest that electron withdrawal at the scissile bond increases k_cat for both NEP and thermolysin. The pH dependence for inhibition by the dipeptides Phe-Ala, Phe-Gly, and Leu-Ala showed binding dependent upon the basic form of an enzyme residue with a pK_a of 7 for NEP and a pK_a of 6 for thermolysin. In the case of thermolysin this pK_a was decreased to 5.3 in the enzymeinhibitor complex. When examined as alternate substrate inhibitors of NEP, N-acyl dipeptides showed three distinct profiles for the dependence of K_i on pH. With N-trifluoroacetyl-Phe-Gly as inhibitor, binding is dependent upon the basic form of an enzyme residue with a pK_a value of 6.2. N-methoxyacetyl-Phe-Gly inhibition appears pH independent, while N-acetyl-Phe-Gly inhibition is dependent upon the acidic form of an enzyme residue with a pK_a of approximately 7. All inhibitions of thermolysin by N-acyl dipeptides exhibit a dependence on the acidic form of an enzyme residue with a pK_a of 5.3 to 5.8. These results suggest that with NEP, binding interactions at the active site involve one or more histidine residues while with thermolysin binding involves an active site glutamic acid residue.