Beta-secondary and solvent deuterium kinetic isotope effects have been determined 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 the class C beta-lactamase of Enterobacter cloacae P99. Although these enzymes are evolutionarily related and have very similar tertiary and active site structure, they are functionally very different-the former efficiently catalyzes the hydrolysis of beta-lactams but not acyclic peptides while vice versa applies to the latter. The measured kinetic isotope effects reveal both similarities and differences in the steady-state transition states for turnover of the various substrates by these enzymes. In most cases, inverse beta-secondary isotope effects were observed, reflecting typical acyl-transfer transition states. With one substrate, however, m-[[(phenylacetyl)glycyl]oxy]benzoic acid, isotope effects on V/K of very close to unity were obtained for both enzymes. These were interpreted in terms of acylation transition state conformations where the extent of beta-CH hyperconjugation was similar 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 isotope effects on V/K were uniformly small, some even inverse, for both enzymes and with all substrates tested. At face value, this suggests the counterintuitive conclusion that little proton transfer occurs in acylation transition states in all of these instances. Closer analysis, however, suggests that for ester and amide (and probably beta-lactam) substrates, this result probably arises from an increase in proton fractionation factors on substrate binding being offset by their decrease in the acylation transition state. The former event derives from proton rearrangement on substrate binding and the latter, presumably, from general acid/base catalysis. This result may be general to all beta-lactam-recognizing enzymes. The solvent isotope effects 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 tetrahedral intermediate where no protonation of the leaving thiolate is required. Deacylation transition states of both enzymes appear to involve significant proton transfer, presumably arising from general acid/base catalysis.