Biochemical characterization of an enzymatic amadori rearrangement in histidine biosynthesis
Abstract
The fourth step in histidine biosynthesis is catalyzed by the hisA gene product which encodes the 5$\prime$-ProFAR isomerase. A detailed investigation was initiated in an effort to better understand this isomerization reaction, the mechanism of catalysis, and the protein function. Recombinant DNA techniques were utilized to facilitate the expression and purification of milligram quantities of protein. An in vitro enzymatic synthesis for substrate and a coupled enzyme assay enabled the evaluation of the basic characteristics of the enzyme. The role of the highly conserved histidine 47 was explored by site-directed mutagenesis. The mutant was purified to homogeneity and determined to be ten fold less efficient than wild type. The relatively high activity of this mutant implies that it is not an obligatory player in catalysis, but a role in substrate binding can not be excluded. An arginine selective modifying reagent, phenylglyoxal, was used to investigate the involvement of these residues. A kinetic evaluation of the inactivation process suggests that a single amino acid residue per enzyme active site is sufficient for inactivation. A series of site-directed mutagenesis has been conducted with the intent of defining the roles of three conserved basic residues. To ascertain the necessity of the anionic phosphate moieties on binding and catalysis, the nucleoside derivatives of the substrate were prepared. None were substrates, and they demonstrate a 20 to 100 fold decrease in affinity. The lack of a primary kinetic isotope using ($\sp2$H-2$\prime\prime$) 5$\prime$-ProFAR implies that proton abstraction at C-2$\prime\prime$ is not the rate determining step. A very large solvent isotope effect is observed. A kinetic evaluation of the solvent isotope effect has led to the discovery that a step after catalysis is limiting, and a proton inventory has suggested that a single proton is involved in this step.
Degree
Ph.D.
Advisors
Davisson, Purdue University.
Subject Area
Biochemistry|Molecular biology
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