Abstract
Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine 153 results in a mutant enzyme with diminished catalytic activity, but with K(m) values for malate and oxaloacetate that are surprisingly unaffected. Reaction 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 site of malate dehydrogenase. The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity for several inhibitors over that observed with the native enzyme. However, the recovery of catalytic activity is less dramatic, with less than one third of the native activity achieved with the optimal reagents. These modified enzymes do have altered substrate specificity, with alpha-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.
Highlights
Malate dehydrogenase from Escherichia coli1 catalyzes the interconversion of malate and oxaloacetate, a critical step in carbohydrate metabolism
In this report we have examined the effect of introducing unnatural amino acids at a single active site position on the substrate specificity of eMDH
Characterization of the R153C Mutant—The active site arginine (Arg-153) that interacts with the ␣-carboxyl group of the substrate (Fig. 1) has been replaced with a cysteine by using the recombinant circle-polymerase chain reaction (RC-PCR) method
Summary
Malate dehydrogenase from Escherichia coli (eMDH) catalyzes the interconversion of malate and oxaloacetate, a critical step in carbohydrate metabolism. As expected for the catalysis of this important metabolic reaction, malate dehydrogenase in both prokaryotes and eukaryotes is highly selective for malate and oxaloacetate, with strong discrimination against other 2-keto mono- and dicarboxylic acids [1]. Several attempts at altering the specificity of this highly selective enzyme have been made, with limited success [1, 3]. It appears that significant changes in specificity will require the additional flexibility of chemical modification to incorporate new functional groups into the active site of eMDH. In this report we have examined the effect of introducing unnatural amino acids at a single active site position on the substrate specificity of eMDH
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