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.
Gutheil
W G
WG
Department of Biochemistry, Meharry Medical College, Nashville, Tennessee 37208, USA.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D006602:Hexosyltransferases; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D019344:Lactic Acid / Q000032:analysis*; D007934:Leuconostoc / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000737:chemistry; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis* / Q000378:metabolism*; D009243:NAD; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D046915:Penicillin-Binding Proteins; D009243:NAD; D019344:Lactic Acid; D007770:L-Lactate Dehydrogenase; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase