Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase.
Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III. With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected. In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included. All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.
Johansson
Carina
C
Department of Biochemistry, Göteborg University, Sweden.
Pedersen
Anders
A
Karlsson
B Göran
BG
Rydström
Jan
J
D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D013050:Spectrometry, Fluorescence
D011522:Protons; D009249:NADP; D009250:NADP Transhydrogenases; D003545:Cysteine