Malate dehydrogenase specifically oxidizes malate to oxaloacetate. The specificity arises from three arginines in the active site pocket that coordinate the carboxyl groups of the substrate and stabilize the newly forming hydroxyl/keto group during catalysis. Here, the role of Arg-153 in distinguishing substrate specificity is examd. by the mutant R153C. The x-ray structure of the NAD binary complex at 2.1 .ANG. reveals two sulfate ions bound in the closed form of the active site. The sulfate that occupies the substrate binding site has been translated .apprx.2 .ANG. toward the opening of the active site cavity. Its new location suggests that the low catalytic turnover obsd. in the R153C mutant may be due to misalignment of the hydroxyl or ketone group of the substrate with the appropriate catalytic residues. In the NAD×pyruvate ternary complex, the monocarboxylic inhibitor is bound in the open conformation of the active site. The pyruvate is coordinated not by the active site arginines, but through weak hydrogen bonds to the amide backbone. Energy minimized mol. models of unnatural analogs of R153C reveal that the regenerated amino and amido side chains can form favorable hydrogen-bonding interactions with the substrate, although a return to native enzymic activity is not obsd. The low activity of the modified R153C enzymes suggests that precise positioning of the guanidino side chain is essential for optimal orientation of the substrate.