Alteration of the specificity of malate dehydrogenase by chemical modulation of an active site arginine

Malate dehydrogenase from Escherichia coli is highly specific for the oxida tion of malate to oxaloacetate. The technique of site-specific modulation h as been used to alter the substrate binding site of this enzyme. Introducti on of a cysteine in place of the active site binding residue arginine 153 r esults in a mutant enzyme with diminished catalytic activity, but with K-m values for malate and oxaloacetate that are surprisingly unaffected. Reacti on of this introduced cysteine with a series of amino acid analog reagents leads to the incorporation of a range of functional groups at the active si te of malate dehydrogenase. The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinit y for several inhibitors over that observed with the native enzyme. However , the recovery of catalytic activity is less dramatic, with less than one t hird of the native activity achieved with the optimal reagents. These modif ied enzymes do have altered substrate specificity, with a-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.