Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.
Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.
Williams
LaKenya
L
Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77842-3012, USA.
Nguyen
Tinh
T
Li
Yingchun
Y
Porter
Tamiko N
TN
Raushel
Frank M
FM
D019747:Aldose-Ketose Isomerases; D000581:Amidohydrolases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D002614:Chelating Agents / Q000494:pharmacology; D003956:Dialysis; D006859:Hydrogen; D007700:Kinetics; D008670:Metals / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010848:Picolinic Acids / Q000494:pharmacology; D011522:Protons; D015032:Zinc / Q000032:analysis
D002413:Cations, Divalent; D002614:Chelating Agents; D008670:Metals; D010848:Picolinic Acids; D011522:Protons; D006859:Hydrogen; D000581:Amidohydrolases; D019747:Aldose-Ketose Isomerases; D015032:Zinc; C004860:dipicolinic acid