期刊
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
卷 77, 期 2, 页码 268-278出版社
WILEY
DOI: 10.1002/prot.22433
关键词
enzyme kinetics; chimera; coenzyme specificity; glutamate dehydrogenase; positive cooperativity
资金
- Enterprise Ireland
- Science Foundation Ireland
Domain-swopped chimeras of the glutamate dehydrogenases from Clostridium symbiosum (CsGDH) (NAD+-specific) and Escherichia coli (EcGDH) (NADP+-specific) have been produced, with the aim of testing the localization of determinants of coenzyme specificity. An active chimera consisting of the substrate-binding domain (Domain 1) of CsGDH and the coenzyme-binding domain (Domain II) of EcGDH has been purified to homogeneity, and a thorough kinetic analysis has been carried out. Results indicate that selectivity for the phosphorylated coenzyme does indeed reside solely in Domain 11; the chimera utilizes NAD(+) at 0.8% of the rate observed with NADP(+), similar to the 0.5% ratio for EcGDH. Positive cooperativity toward L-glutamate, characteristic of CsGDH, has been retained with Domain 1. An unforeseen feature of this chimera, however, is that, although glutamate cooperativity occurs only at higher PH values in the parent CsGDH, the chimeric protein shows it over the full pH range explored. Also surprising is that the chimera is capable of catalysing severalfold higher reaction rates (V-max) in both directions than either of the parent enzymes from which it is constructed. Proteins 2009; 77:268-278. (C) 2009 Wiley-Liss, Inc.
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