The mechanisms of phosphotransferases, nucleotidyltransferases and ATP-dependent synthetases will be characterized with reference to the question of whether substitution at phosphorus is mediated by the transient formation of covalent phosyphorylor nucleotidyl-enzymes as compulsory intermediates. To this end the stereochemical courses of substitution at phosphorus will be established. These enzymes include gentamicin, nucleotidyltransferase, galactokinase, nucleoside phosphotransferase, aminoimidazole ribotide synthetase and glycinamide ribotide synthetase among others. The phosphorus-chiral molecules (R-p)- or (S-p)-[alpha-17-0,18-0]ATP,[gamma-18-0]ATP-gamma-S and [gamma-17-0,18-0]ATP gamma S will be applied in these studies, as well as (R-p)- and (S-p)-[beta-18-0] UDPbetaS-galactose. The interactions of the sulfur-bridging analog of pyrophosphate (PSPi) with a number of phosphotransferases, phosphohydrolases and ATP-dependent synthetases will be investigated with reference to the substrate and inhibitory properties of this species. The nonenzymatic hydrolysis of PSPi and MgPSPi will also be quantitively compared with those of PPi and MgPPi. Synthesis of sulfur-bridging analogs of nucleotides, such as AMPPSP, AMPSPP, and UMPSPGIc, will be undertaken once the properties of synthetic model compounds, such as p1,p2-diethyl-PSP and ethyl-PSPP, have been delineated. Methods for carrying out the syntheses of these unknown molecules will be developed. The active sites and reaction mechanisms of galactose-1-P uridylyltransferase and UDP-galactose 4-epimerase will be characterized in experiments designed to identify active site functional groups that directly catalyze the reactions. Specific chemical modification using affinity labeling agents and radio-chemically labeled substrates will be applied, as well as site-directed mutagenesis. The catalytic roles of these functional groups will be rigorously defined.
| McCoy, Jason G; Arabshahi, Abolfazl; Bitto, Eduard et al. (2006) Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana. Biochemistry 45:3154-62 |
