| Abstract
| - β-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 ofStreptomyces R61 and the class C β-lactamase of Enterobacter cloacae P99. Although these enzymes areevolutionarily related and have very similar tertiary and active site structure, they are functionally verydifferentthe former efficiently catalyzes the hydrolysis of β-lactams but not acyclic peptides while viceversa applies to the latter. The measured kinetic isotope effects reveal both similarities and differences inthe steady-state transition states for turnover of the various substrates by these enzymes. In most cases,inverse β-secondary isotope effects were observed, reflecting typical acyl-transfer transition states. Withone substrate, however, m-[[(phenylacetyl)glycyl]oxy]benzoic acid, isotope effects on V/K of very closeto unity were obtained for both enzymes. These were interpreted in terms of acylation transition stateconformations where the extent of β-CH hyperconjugation was similar to that in the free substrate.Differences in deacylation transition states (V) between the two enzymes with this substrate were interpretedin terms of different acyl-enzyme conformations. Solvent deuterium kinetic isotope effects on V/K wereuniformly small, some even inverse, for both enzymes and with all substrates tested. At face value, thissuggests the counterintuitive conclusion that little proton transfer occurs in acylation transition states inall of these instances. Closer analysis, however, suggests that for ester and amide (and probably β-lactam)substrates, this result probably arises from an increase in proton fractionation factors on substrate bindingbeing offset by their decrease in the acylation transition state. The former event derives from protonrearrangement on substrate binding and the latter, presumably, from general acid/base catalysis. Thisresult may be general to all β-lactam-recognizing enzymes. The solvent isotope effects also suggest that,at least for the P99 β-lactamase, the acylation transition state of a thioester substrate does not involveproton transfer. This can be interpreted in terms of the rate-determining breakdown of a tetrahedralintermediate where no protonation of the leaving thiolate is required. Deacylation transition states of bothenzymes appear to involve significant proton transfer, presumably arising from general acid/base catalysis.
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