Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated with respect to the roles of its cysteine residues. This enzyme contains seven cysteines, of which five are located in the alpha subunit and two are in the beta subunit. All cysteines were replaced by site-directed mutagenesis. The final construct (alphaC292T, alphaC339T, alphaC395S, alphaC397T, alphaC435S, betaC147S, betaC260S) was inserted normally in the membrane and underwent the normal NADPH-dependent conformational change of the beta subunit to a trypsin-sensitive state. Reduction of NADP+ by NADH driven by ATP hydrolysis or respiration was between 32% and 65% of the corresponding wild-type activities. Likewise, the catalytic and proton pumping activities of the purified cysteine-free enzyme were at least 30% of the purified wild-type enzyme activities. The H+/H- ratio for both enzymes was 0.5, although the cysteine-free enzyme appeared to be more stable than the wild-type enzyme in proteoliposomes. No bound NADP(H) was detected in the enzymes. Modification of transhydrogenase by diethyl pyrocarbonate and the subsequent inhibition of the enzyme were unaffected by removal of the cysteines, indicating a lack of involvement of cysteines in this process. Replacement of cysteine residues in the alpha subunit resulted in no or little change in activity, suggesting that the basis for the decreased activity was probably the modification of the conserved beta-subunit residue Cys-260 or (less likely) the non-conserved beta-subunit residue Cys-147. It is concluded that the cysteine-free transhydrogenase is structurally and mechanistically very similar to the wild-type enzyme, with minor modifications of the properties of the NADP(H) site, possibly mediated by the betaC260S mutation. The cysteine-free construct will be a valuable tool for studying structure-function relationships of transhydrogenases.
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PMID:Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase. 918 34

Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the alpha and the beta subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase. Despite harsh treatments with, e.g. detergents and denaturing agents, the alpha and beta subunits remained tightly associated. A monoclonal antibody directed towards the alpha subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II. Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions.
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PMID:Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli. Effects on catalytic and H+-pumping activities. 1082 14