Significant contribution of arginine-112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin. 1994

K Nakamura, and T Horiuchi, and T Yasukochi, and K Sekimizu, and T Hara, and Y Sagara
Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

Cytochrome P-450cam of Pseudomonas putida is a prototype of various eukaryotic cytochrome P-450 molecules. Arg-112 located on the surface of this protein is highly conserved among various other cytochromes P-450. In this study, we constructed mutant genes for P-450cam in which Arg-112 was replaced by Gln or Glu, expressed them in Escherichia coli and purified the mutant proteins. Their enzymic activities were analyzed in the reconstituted system to determine the function of Arg-112. Kd values for d-camphor of Arg112-Gln and Arg112-Glu were much the same as those of the wild-type enzyme, whereas Kd values for the oxidized form of putidaredoxin, which is an acidic protein and is the redox partner of P-450cam, were 240 and 530 microM, respectively. These values are 8 and 19 times larger than that of the wild-type enzyme (28 microM), thereby indicating lower affinities of the mutant enzymes for the oxidized putidaredoxin. Reaction rate constants for reduction by the reduced form of putidaredoxin, measured using the stopped flow method, were 45.5, 9.0 x 10(-3) and 9.0 x 10(-4) s-1 for the wild type, Arg112-Gln and Arg112-Glu, respectively. Thus, Arg-112 of P-450cam plays an important role in the interaction with putidaredoxin and in the high efficiency of the electron transfer; the positive charge of the residue seeming to contribute to the process. The yields in Escherichia coli, the heme contents in the purified fractions and heat stability of the mutant proteins were lower than those of the wild type enzyme, suggesting that Arg-112 of P-450cam is also important for stability of P-450cam.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D002164 Camphor A bicyclic monoterpene ketone found widely in plants, especially CINNAMOMUM CAMPHORA. It is used topically as a skin antipruritic and as an anti-infective agent. Camphor, (+-)-Isomer,Camphor, (1R)-Isomer,Camphor, (1S)-Isomer
D003577 Cytochrome P-450 Enzyme System A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism. Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D004579 Electron Transport The process by which ELECTRONS are transported from a reduced substrate to molecular OXYGEN. (From Bennington, Saunders Dictionary and Encyclopedia of Laboratory Medicine and Technology, 1984, p270) Respiratory Chain,Chain, Respiratory,Chains, Respiratory,Respiratory Chains,Transport, Electron
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005288 Ferredoxins Iron-containing proteins that transfer electrons, usually at a low potential, to flavoproteins; the iron is not present as in heme. (McGraw-Hill Dictionary of Scientific and Technical Terms, 5th ed) Ferredoxin,Ferredoxin I,Ferredoxin II,Ferredoxin III
D006418 Heme The color-furnishing portion of hemoglobin. It is found free in tissues and as the prosthetic group in many hemeproteins. Ferroprotoporphyrin,Protoheme,Haem,Heme b,Protoheme IX
D001120 Arginine An essential amino acid that is physiologically active in the L-form. Arginine Hydrochloride,Arginine, L-Isomer,DL-Arginine Acetate, Monohydrate,L-Arginine,Arginine, L Isomer,DL Arginine Acetate, Monohydrate,Hydrochloride, Arginine,L Arginine,L-Isomer Arginine,Monohydrate DL-Arginine Acetate

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