Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI.
HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Weijun
W
Wang
Stephen Y K
SY
Seah
D000409:Alanine / Q000235:genetics; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry; D019216:Metals, Heavy / Q000737:chemistry; D010648:Phenylacetates / Q000737:chemistry / Q000378:metabolism; D019289:Pyruvic Acid / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D019216:Metals, Heavy; D010648:Phenylacetates; C026246:4-hydroxyphenylacetate; D019289:Pyruvic Acid; D001120:Arginine; D008291:Malate Dehydrogenase; C115453:4-hydroxy-2-ketopentanoic acid aldolase; D000446:Aldehyde-Lyases; C503556:HpaI protein, E coli; C413570:2-dehydro-3-deoxygalactarate aldolase; D000409:Alanine