The effects of mutations at position 253 on the thermostability of the Bacillus subtilis 3-isopropylmalate dehydrogenase subunit interface.
3-Isopropylmalate dehydrogenase (IPMDH) is a dimeric enzyme with a strongly hydrophobic core that is composed of residues from four alpha-helices. We replaced Glu253, which is found in the hydrophobic core and is part of the subunit interface of the Bacillus subtilis (Bs) IPMDH, with several other amino acids to probe. The thermostabilities of the mutants were assessed by measuring the residual enzymatic activities at 40 degrees C after heat treatment and by monitoring changes in ellipticity at 222 nm as the environmental temperature increased incrementally. The results of these studies indicate that, for residues with non-polar side chains, when positioned at residue 253, the thermostabilities of their corresponding mutants correlate positively with the relative hydrophobicities of the side chains. Relative activities of all mutants are lower than that of the wild-type enzyme. For two of the mutants, we directly show that the substitution at position 253 negatively affects Mn(2+) binding, which is required for catalysis. When a lysine is the position 253 residue, the protein dissociates. The results presented herein increase our understanding of the role played by the BsIPMDH dimer interface on the stability and activity of BsIPMDH.
Ohkuri
Takatoshi
T
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo, Japan.
Yamagishi
Akihiko
A
D050539:3-Isopropylmalate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D004777:Environment; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008968:Molecular Conformation; D009154:Mutation; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology
D004798:Enzymes; D015636:Magnesium Chloride; D050539:3-Isopropylmalate Dehydrogenase