Modular coenzyme specificity: a domain-swopped chimera of glutamate dehydrogenase.
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 I) 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 II; 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 I. 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.
Sharkey
Michael A
MA
UCD School of Biomolecular and Biomedical Sciences, Conway Institute, University College Dublin, Belfield, Dublin 4, Republic of Ireland. paul.engel@ucd.ie
Engel
Paul C
PC
D048009:Clostridium symbiosum / Q000201:enzymology*; D003067:Coenzymes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003067:Coenzymes; D011993:Recombinant Fusion Proteins; D009243:NAD; D009249:NADP; D005969:Glutamate Dehydrogenase