Structural basis of substrate specificity in malate dehydrogenases:: Crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, α-ketomalonate and tetrahydoNAD

被引:85
作者
Chapman, ADM
Cortés, A
Dafforn, TR
Clarke, AR
Brady, RL [1 ]
机构
[1] Univ Bristol, Dept Biochem, Bristol BS8 1TD, Avon, England
[2] Univ Barcelona, Dept Biochem & Mol Biol, Fac Chem, Barcelona 08028, Spain
[3] Univ Cambridge, MRC Ctr, Dept Haematol, Cambridge CB4 2QH, England
基金
英国生物技术与生命科学研究理事会;
关键词
malate dehydrogenase; protein crystallography; active site loop; ternary complex; reaction mechanism;
D O I
10.1006/jmbi.1998.2357
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The structural basis for the extreme discrimination achieved by malate dehydrogenases between a variety of closely related substrates encountered within the cell has been difficult to assess because of the lack of an appropriate catalytically competent structure of the enzyme. Here, we have determined the crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase with the alternative substrate alpha-ketomalonate and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide. Both subunits of the dimeric porcine cytoplasmic malate dehydrogenase have a very similar overall topology to previously reported forms of malate dehydrogenase from porcine heart, and from the prokaryotes Escherichia coli and Thermus flavus. However, large changes are noted around the active site, where a mobile loop now closes to bring key residues into contact with the substrate. This observation substantiates a postulated mechanism in which the enzyme achieves high levels of substrate discrimination through charge balancing in the active site. As the activated cofactor/substrate complex has a net negative charge, a positive counter-charge is provided by a conserved arginine in the active site loop. The enzyme must, however, also discriminate against smaller substrates, such as pyruvate. The structure shows in the closed (loop down) catalytically competent complex two arginine residues (91 and 97) are driven into close proximity. Without the complimentary, negative charge of the substrate side-chain of oxaloacetate or alpha-ketomalonate, charge repulsion would resist formation production of this catalytically productive conformation, hence minimising the effectiveness of pyruvate as a substrate. By this mechanism, malate dehydrogenase uses charge balancing to achieve fivefold orders of magnitude in discrimination between potential substrates. (C) 1999 Academic Press.
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页码:703 / 712
页数:10
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