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Science 16 December 1988:
Vol. 242. no. 4885, pp. 1541 - 1544
DOI: 10.1126/science.3201242
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Articles

Science, Vol 242, Issue 4885, 1541-1544
Copyright © 1988 by American Association for the Advancement of Science

articles

A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework

HM Wilks, KW Hart, R Feeney, CR Dunn, H Muirhead, WN Chia, DA Barstow, T Atkinson, AR Clarke, and JJ Holbrook

Department of Biochemistry, University of Bristol, United Kingdom.

Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.


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