It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.

Carbamoyl phosphate synthetase catalyzes the hydrolysis of glutamine by the nucleophilic attack of an active site cysteine residue through a mechanism that requires the formation of a gamma-glutamyl thioester intermediate. The steady-state mole fraction of the thioester intermediate was determined to be 0.23 in the presence and absence of ATP and bicarbonate. The kinetics of formation and hydrolysis of the gamma-glutamyl thioester intermediate during CPS catalyzed hydrolysis of glutamine were determined. When ATP and bicarbonate are added to CPS and glutamine, the kcat for glutamine hydrolysis increases from 0.17 to 150 min-1. The observed rate constant for thioester intermediate formation increases from 18 to 580 min-1, and the microscopic rate constant for hydrolysis of the intermediate increases from 0.15 to 460 min-1. These results demonstrate the kinetic competence of the thioester intermediate during glutamine hydrolysis. The rate-determining step changes from the hydrolysis of the intermediate when ATP and bicarbonate are absent to the formation of the intermediate upon the addition of ATP and bicarbonate. The 3 order of magnitude increase in the rate of glutamine hydrolysis upon the addition of ATP and bicarbonate is indicative of the allosteric communication between two of the three reaction centers of CPS. These sites are physically separated by approximately 45 A.

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Five alkynyl phosphate esters have been synthesized as probes of the active site structure of phosphotriesterase. These compounds have the potential to be converted by the enzyme to a highly reactive ketene intermediate which can then react with an active site nucleophile causing irreversible inhibition of the enzyme by formation of an inactive covalent adduct. All five compounds completely inactivate enzyme function in less than 15 s at pH 7.0. The partition rations of 1-hexynyl diethyl phosphate (I), 1-propynyl diethyl phosphate (II), 1-hexynyl diphenyl phosphate (III), 1-hexynyl dimethyl phosphate (IV), and ethynyl diethyl phosphate (V) fall in the range between 480 and 1700; thus, all five alkynyl phosphate esters work equally well as inactivators despite the differences in their structures. The rate constants for enzyme inactivation, kinact, are 1.7 s-1 with I, 1.3 s-1 with II, and 0.12 s-1 with IV. They compare well with the kcat for the Co-substituted phosphotriesterase; hence these compounds are good substrates. The stoichiometry of inhibitor bound to protein is 1:1, as determined by inactivation of the enzyme using the radiolabeled compound [3-14C]-1-propynyl diethyl phosphate. Addition of an exogenous nucleophile, azide, did not protect phosphotriesterase from being inactivated by the alkynyl phosphate esters, suggesting that the reactive intermediate produced from the inhibitor is not released from the enzyme surface prior to covalent labeling of the protein. Chemical and spectroscopic evidence suggests that a histidine residue is modified in the inactivation reaction. The inactivated phosphotriesterase can be reactivated by increasing the pH of the protein solution. N-Acylimidazoles are known to be easily hydrolyzed at alkaline pH values.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoplasmic fractions from species of the Mollicutes genera Entomoplusma, Mesoplasma, Mycoplusma, and Acholeplusma were assayed for NADH oxidase (NADH ox), ATP-and PP,-dependent phosphofructokinase (PFK), ATP-and PP,-dependent deoxyguanosine kinase (dGUOK), thymidine kinase (TK), TMP kinase (TMPK), glucose-6-phosphate dehydrogenase (MPde), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), phosphoenolpyruvate carboxylase, hypoxanthine-guanine phosphoribosyl transferase, dUTPase, and uracil-DNA glycosylase (UNG) activities. Membrane fractions were also examined for NADH ox activity. These activities were used as indicators of the presence and relative activities of major MoZZicutes metabolic and DNA repair pathways. This was the first study to determine the presence of these enzymes in members of the genera Entomoplusma and Mesoplusma. Using the data obtained, we constructed a preliminary scheme for distinguishing genera of the class Mollicutes on the basis of the results of signature functional enzyme assays. This scheme includes phylogenetic relationships deduced from rRNA analyses, but is more informative with respect to metabolic potential. The criteria used include the presence of PP,-dependent PFK, urease, dUTPase, and dGUOK activities. Entomoplasma ellychniae ELCN-lT (T = type strain), Entomoplasma meluleucae M-lT, Mesoplasma segertii F7T, Mesoplusma entomophilum TACT, Mesoplusma jlorum LIT, Mycoplusma fermentans PGIST, and AchoZeplasma multibcale PN525T were similar in most respects. NADH ox activity was localized in the cytoplasm of these organisms. These strains had ATP-dependent PFK, MDH, LDH, ATP-and PP,-dependent dGUOK, and UNG activities, but not dUTPase or G6Pde activities. In contrast, Acholeplusma equifetale C112T, Acholeplasma oculi 19LT, Acholeplasma hippikon CIT, Acholeplusma modicum PG49T, and Acholeplasma momm 72-043T had membrane-localized NADH ox activity, PP,-dependent PFK, G6Pde, and dUTPase activities, and significantly lower MDH and LDH activities and exhibited a faster rate with PP, than with ATP in the dGUOK reaction. All of the members of the MoZZicutes tested had hypoxanthine-guanine phosphoribosyl transferase, phosphoenolpyruvate carboxylase, and (except for Mesoplasma entomophilum TACT) UNG activities. All of the Acholeplusma strains except Acholeplusmu multilocale PN52ST had TK, TMPK, and UNG activities. Mesoplusma entomophilum TACT was distinguished by having no detectable dUTPase, UNG, TK, and TMPK activities, indicating that there is a severe restriction in or an absence of a synthetic route to dTTP. Our data also suggest that A. multiZocale PN52ST is a member of an unrecognized metabolic subgroup of the genus Acholeplusm or is not an Acholeplusma strain.Distinguishing members of the class Mollicutes by their metabolic characteristics has been of very limited phylogenetic and taxonomic usefulness. Standards for describing Mollicutes taxa have been published previously (7). Characteristics such as gross cellular morphology, colonial appearance, genome size, and serologic...

Bacterial alkaline phosphatase catalyzes the hydrolysis and transphosphorylation of phosphate monoesters. Site-directed mutagenesis was used to change the active-site residue Asp-153 to Ala and Asn. In the wild-type enzyme Asp-153 forms a second-sphere complex with Mg2+. The activity of mutant enzymes D153N and D153A is dependent on the inclusion of Mg2+ in the assay buffer. The steady-state kinetic parameters of the D153N mutant display small enhancements, relative to wild type, in buffers containing 10 mM Mg2+. In contrast, the D153A mutation gives rise to a 6.3-fold increase in kcat, a 13.7-fold increase in kcat/Km (50 mM Tris, pH 8), and a 159-fold increase in Ki for Pi (1 M Tris, pH 8). In addition, the activity of D153A increases 25-fold as the pH is increased from 7 to 9. D153A hydrolyzes substrates with widely differing pKa's of their phenolic leaving groups (PNPP and DNPP), at similar rates. As with wild type, the rate-determining step takes place after the initial nucleophilic displacement (k2). The increase in kcat for the D153A mutant indicates that the rate of release of phosphate from the enzyme product complex (k4) has been enhanced.

The alkynyl phosphate ester, 1-hexynyl diethyl phosphate (I), is a mechanism-based inhibitor of phosphotriesterase. It has been previously determined that a histidine residue in the wild-type phosphotriesterase is covalently modified by this compound. In order to identify which of the seven histidine residues in the native enzyme are required for inactivation, the kinetic properties of phosphotriesterase mutants with this suicide substrate were examined in detail. Six of the seven mutants (histidine to asparagine) were rapidly inactivated by I. The mutants H55N, H57N, and H230N also showed partition ratios that were lower than for the wild-type enzyme. The rate of inactivation of H201N was significantly slower than that of wild-type phosphotriesterase. The H254N mutant could not be inactivated; no more than 60% of the initial activity was lost, even at I/E0 ratios of 4000:1. These results suggest that His-254 is essential for the inactivation of phosphotriesterase and is likely to be the primary target in the wild-type enzyme for modification by I. The inactivation of wild-type phosphotriesterase and the seven mutants was also studied using diethyl pyrocarbonate, a histidine selective reagent. The second-order rate constant for the inactivation of wild-type phosphotriesterase was determined to be 1.3 M-1 min-1. The rate constants for the inactivation of the H55N, H57N, H201N, and H230N mutants were larger than for the wild-type enzyme. Thus, it appears that when these histidine residues are replaced by asparagine, other histidine residues in the active site become more susceptible to modification, resulting in a faster rate of inactivation. The mutant H254N was not inactivated in the presence of DEPC.(ABSTRACT TRUNCATED AT 250 WORDS)

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