Dioxygenases without requirement for cofactors: identification of amino acid residues involved in substrate binding and catalysis, and testing for rate-limiting steps in the reaction of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase.
1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod), catalyzing cleavage of its heteroaromatic substrate to form carbon monoxide and N-acetylanthranilate, belongs to the alpha/beta hydrolase fold family of enzymes. Analysis of protein variants suggested that Hod has adapted active-site residues of the alpha/beta hydrolase fold for the dioxygenolytic reaction. H251 was recently shown to act as a general base to abstract a proton from the organic substrate. Residue S101, which corresponds to the nucleophile of the catalytic triad of alpha/beta-hydrolases, presumably participates in binding the heteroaromatic substrate. H102 and residues located in the topological region of the triad's acidic residue appear to influence O2 binding and reactivity. A tyrosine residue might be involved in the turnover of the ternary complex [HodH+-3,4-dioxyquinaldine dianion-O2]. Absence of viscosity effects and kinetic solvent isotope effects suggests that turnover of the ternary complex, rather than substrate binding, product release, or proton movements, involves the rate-determining step in the reaction catalyzed by Hod.
Frerichs-Deeken
Ursula
U
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
Fetzner
Susanne
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003067:Coenzymes / Q000494:pharmacology*; D049308:Dioxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D014783:Viscosity
D000596:Amino Acids; D003067:Coenzymes; C101932:1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (decyclizing, CO-forming); D049308:Dioxygenases