A sensitive equilibrium-based assay for D-lactate using D-lactate dehydrogenase: application to penicillin-binding protein/DD-carboxypeptidase activity assays.

An assay for D-lactate (D-Lac) is described where D-Lac in the presence of NAD+ is equilibrated to NADH and pyruvate (Pyr) by Leuconostoc mesenteroides D-lactate dehydrogenase (DLDH). This assay was standardized using known concentrations of D-Lac and a linearized form of the equilibrium expression. The assay has a lower limit of about 20 microM D-Lac in a 1 ml assay mixture (20 nmol D-Lac). As a demonstration of this assay method it is used to characterize the hydrolysis of the standard penicillin-binding protein/DD-carboxypeptidase substrate Ac2-L-Lys-D-Ala-D-Lac by penicillin-binding protein 5 from Escherichia coli. The approach adopted here of using an inherently nonlinear response, which however follows precisely determinable physical behavior, has the advantages of providing a wider dynamic range and increased relative precision over analogous linear response-based methods. This approach may be applicable to the development of other enzyme-based assay methods.