Biomaterial Database

Identification of a lysine residue in the NADH-binding site of salicylate hydroxylase from Pseudomonas putida S-1. 1995

K Suzuki, and M Mizuguchi, and T Gomi, and E Itagaki

Department of Chemistry, Faculty of Science, Kanazawa University, Ishikawa.

Salicylate hydroxylase from Pseudomonas putida S-1 was irreversibly inactivated by trinitrobenzenesulfonic acid (TNBS). The reaction was linearly dependent on TNBS concentration and the second-order rate constant was 120 M-1.min-1 for the holoprotein at pH 8.5. Modification of one mole of lysine residue per mole of enzyme caused a large loss of the activity, and the enzyme was no longer able to show NADH-dehydrogenase activity after uncoupling. The presence of NADH, NAD+, ATP, or AMP afforded protection against the inactivation. The enzyme modified at a single lysine residue was isolated by hydrophobic chromatography as an apoprotein form and characterized. It could bind FAD with the same Kd value for that of native apoprotein. The apparent Michaelis constant of the enzyme was increased 13-fold for NADH, but not for salicylate. Vmax for NADH oxidation was decreased to one-fifth of that of the native enzyme. A peptide containing one trinitrophenyl-lysine residue was isolated from the chymotryptic digest of the modified enzyme and its amino acid sequence was determined to be TADVAIAADGIKSSM, which is homologous to the sequence from R-154 to I-168 of salicylate hydroxylase from P. putida PpG7. The lysine in the peptide may represent a basic residue interacting with an anionic group of NADH in the binding site of the enzyme.

UI	MeSH Term	Description	Entries
D008239	Lysine	An essential amino acid. It is often added to animal feed.	Enisyl,L-Lysine,Lysine Acetate,Lysine Hydrochloride,Acetate, Lysine,L Lysine
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
D009243	NAD	A coenzyme composed of ribosylnicotinamide 5'-diphosphate coupled to adenosine 5'-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). (Dorland, 27th ed)	Coenzyme I,DPN,Diphosphopyridine Nucleotide,Nadide,Nicotinamide-Adenine Dinucleotide,Dihydronicotinamide Adenine Dinucleotide,NADH,Adenine Dinucleotide, Dihydronicotinamide,Dinucleotide, Dihydronicotinamide Adenine,Dinucleotide, Nicotinamide-Adenine,Nicotinamide Adenine Dinucleotide,Nucleotide, Diphosphopyridine
D011732	Pyridoxal Phosphate	This is the active form of VITAMIN B 6 serving as a coenzyme for synthesis of amino acids, neurotransmitters (serotonin, norepinephrine), sphingolipids, aminolevulinic acid. During transamination of amino acids, pyridoxal phosphate is transiently converted into pyridoxamine phosphate (PYRIDOXAMINE).	Pyridoxal 5-Phosphate,Pyridoxal-P,Phosphate, Pyridoxal,Pyridoxal 5 Phosphate,Pyridoxal P
D002918	Chymotrypsin	A serine endopeptidase secreted by the pancreas as its zymogen, CHYMOTRYPSINOGEN and carried in the pancreatic juice to the duodenum where it is activated by TRYPSIN. It selectively cleaves aromatic amino acids on the carboxyl side.	Alpha-Chymotrypsin Choay,Alphacutanée,Avazyme
D006899	Mixed Function Oxygenases	Widely distributed enzymes that carry out oxidation-reduction reactions in which one atom of the oxygen molecule is incorporated into the organic substrate; the other oxygen atom is reduced and combined with hydrogen ions to form water. They are also known as monooxygenases or hydroxylases. These reactions require two substrates as reductants for each of the two oxygen atoms. There are different classes of monooxygenases depending on the type of hydrogen-providing cosubstrate (COENZYMES) required in the mixed-function oxidation.	Hydroxylase,Hydroxylases,Mixed Function Oxidase,Mixed Function Oxygenase,Monooxygenase,Monooxygenases,Mixed Function Oxidases,Function Oxidase, Mixed,Function Oxygenase, Mixed,Oxidase, Mixed Function,Oxidases, Mixed Function,Oxygenase, Mixed Function,Oxygenases, Mixed Function
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D014302	Trinitrobenzenesulfonic Acid	A reagent that is used to neutralize peptide terminal amino groups.	Picrylsulfonic Acid,Trinitrobenzene Sulfonate,2,4,6-Trinitrobenzene Sulfonate,Trinitrobenzenesulfonic Acid, Sodium Salt,Sulfonate, Trinitrobenzene
D016958	Pseudomonas putida	A species of gram-negative, aerobic bacteria isolated from soil and water as well as clinical specimens. Occasionally it is an opportunistic pathogen.