Browsing by Author "Paquatte, Olivier"
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Item Chemical modification of salicylate hydroxylase(1985) Paquatte, Olivier; Tu, Shiao-Chun; Kimball, Aubrey P.; Nelson, Daniel A.; Kohn, Harold LewisSalicylate hydroxylase from Pseudomonas cepacia was found to be inactivated by phenyl glyoxal, a reagent specific for arginyl residues. The reaction was found to display pseudo-first order kinetics but was not retarded by the addition of any of the substrates before initiating the inactivation. This suggests that the modification does not take place at the enzyme active site. Salicylate hydroxylase can also be effectively inactivated by di ethyl pyrocarbonate, a reagent highly reactive with histidyl residues. The enzyme inactivation was retarded by the addition of salicylate but not NADH, suggesting the modification was limited to the salicylate substrate site. However, the inactivation of enzyme with an excess amount of di ethyl pyrocarbonate did not display pseudo-first order kinetics, due to the appreciable autodecay of the reagent in an aqueous medium. A new method is described for the determination of both the pseudo-first order rate constant and the second order rate constant for the enzyme inactivation by a reagent such as di ethyl pyrocarbonate, which undergoes exponential decays. The validity of this method has been demonstrated by the successful application to the analysis of results of salicylate hydroxylase and bacterial luciferase inactivations by diethyl pyrocarbonate. The effects of temperature, pH and concentration of phosphate buffer on the stability of this reagent have been examined.Item Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents and by chemical modification(1988) Paquatte, Olivier; Tu, Shiao-Chun; Cox, James R., Jr.; Fox, George E.; Eichberg, Joseph E.; Widger, William R.Previously we have established that an essential cysteinyl group on the [alpha] subunit is at the aldehyde site of ([alpha beta]) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the essential cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the essential cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. [...]