KINETIC-STUDIES OF HEMOPHILUS-INFLUENZAE MALATE-DEHYDROGENASE

被引:11
作者
YOON, H [1 ]
ANDERSON, BM [1 ]
机构
[1] VIRGINIA POLYTECH INST & STATE UNIV, DEPT BIOCHEM & NUTR, BLACKSBURG, VA 24061 USA
关键词
D O I
10.1016/0167-4838(88)90174-4
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
Haemophilus infleunzae malate dehydrogenase ((S)-malate:NAD+ oxidoreductase EC 1.1.1.37) was purified 109-fold with a 26% recovery through a four-step procedure involving salt fractionation, hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of Mr 61,000. Initial velocity studies of all four substrates in the forward and reverse reactions indicated a sequential mechanism for the enzyme. Product and dead-end inhibition studies were consistent with an ordered bi-bi mechansm in which NAD is the first substrate bound to the enzyme and NADH, the second product released. Several analogs of NAD structurally altered in either the pyridine or purine moiety were observed to function as coenzymes in the reaction catalyzed by the purified malate dehydrogenase. Alterations in the purine portion of the dinucleotides had a more pronounced effect on the kinetic parameters observed in malate oxidation. The enzyme was inactivated by incubation with diethylpyrocarbonate, whereas no inactivation was observed with sulfhydryl reagents.
引用
收藏
页码:10 / 18
页数:9
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