beta-secondary and solvent deuterium kinetic isotope effects on catalysis by the Streptomyces R61 DD-peptidase: Comparisons with a structurally similar class C beta-lactamase

beta-Secondary and solvent deuterium kinetic isotope effects have been dete rmined for the steady-state kinetic parameters V/K and V for turnover of a series of acyclic substrates by the DD-peptidase of Streptomyces R61 and th e class C beta-lactamase of Enterobacter cloacae P99. Although these enzyme s are evolutionarily related and have very similar tertiary and active site structure, they are functionally very different-the former efficiently cat alyzes the hydrolysis of beta-lactams but not acyclic peptides while vice v ersa applies to the latter. The measured kinetic isotope effects reveal bot h similarities and differences in the steady-state transition states for tu rnover of the various substrates by these enzymes. In most cases, inverse b eta-secondary isotope effects were observed, reflecting typical acyl-transf er transition states. With one substrate, however, m-[[(phenylacetyl)glycyl ] oxy]benzoic acid, isotope effects on V/K of very close to unity were obta ined for both enzymes. These were interpreted in terms of acylation transit ion state conformations where the extent of beta-CH hyperconjugation was si milar to that in the free substrate. Differences in deacylation transition states (V) between the two enzymes with this substrate were interpreted in terms of different acyl-enzyme conformations. Solvent deuterium kinetic iso tope effects on V/K were uniformly small, some even inverse, for both enzym es and with all substrates tested. At face value, this suggests the counter intuitive conclusion that little proton transfer occurs in acylation transi tion states in all of these instances. Closer analysis, however, suggests t hat for ester and amide land probably beta-lactam) substrates, this result probably arises from an increase in proton fractionation factors on substra te binding being offset by their decrease in the acylation transition state . The former event derives from proton rearrangement on substrate binding a nd the latter, presumably, from general acid/base catalysis. This result ma y be general to all beta-lactam-recognizing enzymes. The solvent isotope ef fects also suggest that, at least for the P99 beta-lactamase, the acylation transition state of a thioester substrate does not involve proton transfer . This can be interpreted in terms of the rate-determining breakdown of a t etrahedral intermediate where no protonation of the leaving thiolate is req uired. Deacylation transition states of both enzymes appear to involve sign ificant proton transfer, presumably arising from general acid/base catalysi s.