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

Abstract

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 p K 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 p K 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 p K a of 7 for NEP and a p K a of 6 for thermolysin. In the case of thermolysin this p K 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 p K 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 p K 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 p K 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.