Fred C. Hartman

Synthesis and characterization of cis- and trans-2,3-epoxybutane-1,4-diol 1,4-bisphosphate, potential affinity labels for enzymes that bind sugar bisphosphates

Abstract cis- and trans-2,3-Epoxybutane-1,4-diol 1,4-bisphosphate, which can be considered reactive analogs of several sugar bisphosphates, have been synthesized in a continuing effort to develop new and diverse affinity labeling reagents for enzymes which bind phosphorylated substrates. cis-2,3-Epoxybutane-1,4-diol was obtained by epoxidation of commercially available cis-2-butene-1,4-diol with m-chloroperbenzoic acid; the trans epoxide was obtained by reduction of 2-butyne-1,4-diol with LiAlH4 followed by epoxidation with m-chloroperbenzoic acid. The diols were phosphorylated with diphenyl chlorophosphate, and the phenyl blocking groups were then removed by Pt-catalyzed hydrogenation. By the criterion of their reaction with the sulfhydryl group of glutathione, the phosphorylated epoxides are 6000 times less electrophilic than the previously described and structurally similar reagent 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

Attempts to Apply Affinity Labeling Techniques to Ribulose Bisphosphate Carboxylase/Oxygenase

Although its results are subject to uncertainties in interpretation, chemical modification is a proven method for defining structure-function relationships in enzymes. Innumerable times, tentative conclusions based on chemical modification studies regarding identities of active-site residues, or even their precise catalytic function, have been substantiated by x-ray crystallography.

Detection of haloacetyl and haloketone affinity labels on paper chromatograms

Abstract Thiol-reactive substances on chromatograms can be detected conveniently with a single spray reagent composed of Ellmans reagent and reduced glutathione, Data pertaining to the visualization of reactive halogen compounds are presented.

Function of Active-Site Residues of Ribulose-Bisphosphate Carboxylase/Oxygenase (Rubisco)

Chemical modification [1], site-directed mutagenesis [2–5], and X-ray crystallography [6–11] have identified Lys166, Lys329, and Glu48 as active-site residues of the homodimeric Rubisco from Rhodospirillum rubrum. (In the L8S8 Rubisco from spinach, these residues correspond to Lys175, Lys334, and Glu60.) We have used two strategies to explore the function of these residues: (a) introduction of very subtle structural changes into their side chains by combining chemical modification with site-directed mutagenesis and (b) evaluation of the ability of mutant proteins, devoid in overall carboxylase activity, to catalyze the enolization of D-ribulose-l,5-bisphosphate (RuBP), i.e. the first step in the normal reaction pathway.