STRINGENCY OF SUBSTRATE-SPECIFICITY OF ESCHERICHIA-COLI MALATE-DEHYDROGENASE

Malate dehydrogenase and lactate dehydrogenase are members of the stru cturally and functionally homologous family of 2-ketoacid dehydrogenas es. Both enzymes display high specificity for their respective keto su bstrates, oxaloacetate and pyruvate. Closer analysis of their specific ity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. S ite-specific mutagenesis of the two enzymes in an attempt to reverse t heir specificity has met with contrary results. Conversion of a specif ic active-site glutamine to arginine in lactate dehydrogenase from Bac illus stearothermophilus generated an enzyme that displayed activity t oward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructe d a series of mutants in the mobile, active site loop of the Escherich ia coli malate dehydrogenase that incorporate the complementary change , conversion of arginine 81 to glutamine, to evaluate the role of char ge distribution and conformational flexibility within this loop in def ining the substrate specificity of these enzymes. Mutants incorporatin g the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-spec ificity mutants were much less active than the native enzyme. Secondar y mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M8 5E/D86T) had either no or only moderately beneficial effects on the ac tivity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestl y improved activity toward pyruvate. The possible physiological releva nce of the stringent specificity of malate dehydrogenase was investiga ted. In normal strains off. coli, fermentative metabolism was not affe cted by expression of the mutant malate dehydrogenase. However, when e xpressed in a strain of E. coli unable to ferment glucose, the mutant enzyme restored growth and produced lactic acid as the sole fermentati on product. (C) 1995 Academic Press, Inc.