Program

Vincent Purpero, Kayunta Johnson-Winters, Mike Kavana and GRAHAM R. MORAN. 

University of Wisconsin - Milwaukee. 3210 N. Cramer st, Milwaukee, WI 53211. 

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4- hydroxyphenylpyruvate to homogentisate a reaction that involves decarboxylation, substituent migration and aromatic oxygenation. We have examined both the binding of ligands and oxygen reactivity of HPPD from Streptomyces avermitilis. The binding of either 4-hydroxyphenylpyruvate, phenylpyruvate or pyruvate to the holo-enzyme produces a weak ligand charge transfer band at --500 nm that is indicative of bidentate binding of the 1-carboxylate and 2-keto pyruvate oxygen atoms to the active site metal ion. Interestingly, no turnover is observed in the presence of phenylpyruvate or pyruvate. EPR and MCD of these holo-enzyme complexes suggest a geometric switch that raises the reactivity of the enzyme toward molecular oxygen only in the presence of 4- hydroxyphenylpyruvate. The rate of reduction of molecular oxygen increases 3,600 fold when holo-HPPD is in complex with this substrate. This complex reacts with molecular oxygen inducing the formation of a spectrophotometrically observable intermediates that form and decay at the catalytically relevant rates. 

Specific di and triketones are potent inhibitors of HPPD. Commercially these molecules are used primarily as herbicides that stem the production of quinone redox cofactors required in photosynthesis. However, they also function as therapeutics that completely alleviate the lethal effects of type 1 tyrosinemia. We have examined the mechanism of acquisition of such an inhibitor, NTBC, by HPPD. The mechanism involves three steps, two of which are apparently irreversible and result in ligand charge- transfer at the metal ion. One of the pivotal aspects in relation to inhibition is the complete suppression of the oxygen reactivity in the HPPD.NTBC complex. 

MAX O. FUNK, JR., Ardeschir Vahedi-Faridi, Pierre-Alexandre Brault, Priya Shah, W. Richard Dunham, and Yong-Wah Kim; 

Department of Chemistry, University of Toledo, 2801 West Bancroft Street, Toledo, Ohio, 43606. 

Lipoxygenase catalysis depends in a critical fashion on the redox properties of a mononuclear non-heme iron cofactor. The isolated enzyme contains predominantly if not exclusively iron(II), but the catalytically active form of the enzyme has iron(III). The activating oxidation of the iron takes place in a reaction with the hydroperoxide product of the catalyzed reaction. In a second peroxide dependent process, lipoxygenases are also inactivated. The molecular basis for the inactivation was thought to involve reactions at sensitive amino acid side chains, e.g. methionine, but site-directed mutagenesis To examine the redox experiments failed to identify the susceptible residues. activation/inactivation dichotomy in lipoxygenase chemistry, we investigated the interaction between lipoxygenase-1 (and -3) and cumene hydroperoxide. Cumene hydroperoxide was a reversible inhibitor of the catalyzed reaction under standard assay Reconciliation of the conditions, but strikingly only at high substrate concentrations.Reconciliion of the data with the  ofcu  rrently held kinetic mechanism requires simultaneous binding of substrate and peroxide. It was further found that the enzyme was both oxidized and largely inactivated in a reaction with the peroxide in the absence of substrate. The consequences of this reaction for the enzyme included the B-hydroxylation of amino acid side chains in the vicinity of the cofactor. The modifications were identified by mass spectrometry and X-ray crystallography. The oxidation is accompanied by a subtle rearrangement in the coordination sphere of the iron atom. Since the enzyme retains catalytic activity, albeit diminished, after treatment with cumene hydroperoxide, the structure of the iron(III) site may reflect the catalytically relevant form of the cofactor. 

MARVIN W. MAKINEN', Hesheng Ou2, Devkumar Mustafi', and Matthew J. Brady2 

'Department of Biochemistry & Molecular Biology and Department of Medicine, Section on Endocrinology, The University of Chicago, Chicago, Illinois 60637 

The insulin-mimetic activity of the vanadyl cation (VO2+) increasing glucose uptake in insulin-sensitive cells and tissues is well known (1). We have demonstrated that bis(acetylaceto-nato)oxovanadium(IV) [VO(acac)2] enhances the uptake of 2-deoxy-[1- 14C]-glucose by differ-entiated, serum-starved 3T3-LI adipocytes upon formation of a 1:1 adduct with bovine serum albumin (BSA) (2). To identify target enzymes of VO2+- chelates regulating insulin-signaling pathways, we have compared the metabolic actions of VOSO4 and VO(acac), in 3T3-L1 adipo-cytes. Treatment of cells with 0.25 mM VO(acac)2 in the presence of 0.25 mM BSA caused a 20-fold increase in glycogen synthesis. In contrast, 0.25 mM VOSO4 caused only a 2-fold increase in glycogen accumulation, compared to a maximal 100-fold enhancement by 10 nM insulin. VO(acac)2 synergistically increased glycogen accumulation stimulated by 0.1 and 1 nM insulin but caused no further increase at maximal insulin concentration. In agreement, phosphorylation of Akt and GSK3 was potentiated by 0.25 mM VO(acac); at submaximal insulin concentrations but showed no further increase at 10 nM insulin. However, activation of glycogen synthase was increased at all insulin concentrations. Treatment of 3T3-L1 adipocytes with VO(acac)2 for 5 min potentiated tyrosine phosphorylation of the insulin receptor (IR) and its polypeptide sub-strate IRS-1 at submaximal insulin concentrations. In contrast, VOSO4 had no effect on the phosphorylation of Akt, GSK3, IR, IRS-1 or activation of glycogen synthase. Not only was the influence of bis(maltolato)oxovanadium(IV) (3) and bis(pyridine-1-oxide-2- thiolato)oxovana-dium(IV) (4) decreased compared with VO(acac)2, but also BSA enhanced the insulin-mimetic activity of all three chelates more than serum transferrin. In contrast to the report that the insulin mimetic action of VO2+ involves only activation of post-receptor signaling pathways (5), these results provide evidence that VO(acac)2 acts by potentiating IR tyrosine phosphorylation. (Supported by NIH DK57599. MJB holds a Career Development Award of the American Diabetes Association.) 

REBECCA S. MYERS', Nagarajan Venugopalan2, Janet L. Smith2, V. Jo Davisson' '

Department of Medicinal Chemistry and Molecular Pharmacology, Department of Biological Sciences, Purdue University, West Lafayette, Indiana 

Glutamine amidotransferases (GATs) utilize the amide nitrogen of glutamine to incorporate ammonia into various metabolites. Imidazole glycerol phosphate synthase (IGPS) is a GAT in histidine biosynthesis that catalyzes two carbon-nitrogen ligations with PRFAR and an elimination reaction to form AICAR and IGP. The two active sites in the protein are temporally coupled and reside in distinct domains; one the glutaminase and the second a (B/a), barrel nucleotide binding domain. Our focus has been the protein interactions that signal the glutaminase event when PRFAR is bound.

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Based upon the recent substrate-occupied structure for yeast IGP synthase, kinetic investigations of site- directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction. The binding of the ribulosyl phosphate of PRFAR causes the reorientation of K258 resulting in a conformational change of R239 at the base of the (B/a), core that enables the passage of ammonia through the core of the protein. 

Glutaminase activity is proposed to be stimulated by the nucleotide phosphate binding with T365. This transmits the signal down ẞ4 to D359, a residue that participates in network of hydrogen bonding with N13 and K196, adjacent to the glutaminase active site. D359A resulted in an abolition of PRFAR-stimulated glutaminase activity. N13A reduced this signal 250-fold. Intriguingly, K196A enhanced the PRFAR signal three-fold. A double mutant, D359A/K196A reduced the stimulated glutaminase activity 15-fold, and greatly reduced PRFAR turnover. This mutant may be creating an escape route for ammonia and suggests that there is a secondary pathway for the binding signal between the active sites. 

L. M. WATKINS, R. Rodriguez, D. Schneider, R. Broderick, M. Cruz, R. Chambers, E. Ruckman, J. Dowdy and M. Cody, 

Department of Chemistry and Biochemistry, Texas State University-San Marcos, 601 University Dr., San Marcos, TX 78666 

Dibenzothiophene (DBT) is the model organosulfur compound used to study biodesulfurization of petroleum middle distillate. 2-(2'-hydroxyphenyl)benzenesulfinate desulfinase (HPBS desulfinase) catalyzes the final and rate limiting step in the desulfurization of dibenzothiophene by Rhodococcus erythropolis IGTS8, converting 2- (2'-hydroxyphenyl)benzenesulfinate (HPBS) to 2-hydroxybiphenyl. Sulfite is also formed in the reaction. HPBS desulfinase was purified 1600-fold from Rhodococcus IGTS8. The purification was monitored using a spectrofluorimetric assay and SDS-PAGE. The pl of HPBS desulfinase is 5.6, the temperature optima is 35°C, and the pH optima is 7.0. HPBS desulfinase does not require a cofactor for activity. HPBS desulfinase has a Km of 0.90 ± 0.15 μM and a keat of 1.3 ± 0.07 min. There is an acidic and a basic group required for activity. HPBS desulfinase activity decreases in the presence of Cu2+ and Zn2*, while no metals significantly enhance enzyme activity. Substrate analogs were synthesized and tested for their ability to act as substrates or inhibitors of HPBS desulfinase. No alternative substrates and very few inhibitors were identified. Spectrofluorimetric assays were used to measure binding constants of the inhibitors. Chemical modification experiments, homology studies and site-directed mutagenesis studies suggest that a highly conserved and unique cysteine residue and a nearby tyrosine residue are an essential active site base and acid, respectively, in the mechanism of this enzyme. 

Maria N. Credi, Paul M. Pagano, & BRUCE A. PALFEY 

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606 

The dihydroorotate dehydrogenases (DHODs) use an FMN prosthetic group to catalyze the conversion of dihydroorotate to orotate, the only redox reaction in pyrimidine biosynthesis. The DHODs from most Gram-negative bacteria and most eukaryotes are Class 2 enzymes, which are membrane-bound and use ubiquinone as the physiological oxidant. We are studying the Class 2 enzymes from E. coli and H, sapiens in order to determine the mechanism used to reduce the flavin. The oxidation of dihydroorotate requires the deprotonation of C5 of dihydroorotate and the transfer of a hydride equivalent from C6 of dihydroorotate to N5 of FMN. These two C-H bonds may be cleaved in a concerted or stepwise fashion. We have studied this in anaerobic stopped- flow experiments in the absence of oxidizing substrates, enabling us to directly measure intrinsic rate constants for flavin reduction using protio- or deutero-dihydroorotate. The kinetic isotope effects obtained with label at C5, C6, or C5 and C6 indicate that the reaction is stepwise, in contradiction to the result obtained for bovine liver DHOD by steady-state kinetics [Hines, V. & Johnston, M. (1989) Biochemistry, 28, 1227-1234]. Two stepwise mechanisms are being considered: α-deprotonation by the base to form an enolate intermediate that reduces the flavin, or ẞ-hydride abstraction by the flavin to form an iminium cation intermediate that loses a proton to the active site serine. (Supported by NIH GM61087

JOO YOUNG CHA*, Michael Norgard**, and Shahriar Mobashery*, *

Dept. of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, **Dept. of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390 

Treponema pallidum is the spirochete bacterium that causes syphilis. T. pallidum is exceptionally sensitive to penicillins, but the lethal targets for these antibiotics are still unknown. The in vitro incubation with radiolabeled B-lactam reveals that T. pallidum encodes several PBPs, an assertion supported by the genome sequence of the organism. It was determined that a major PBP is a 47-kDa lipoprotein (Tp47). This protein also exhibits a novel B-lactamase activity, which would appear to operate by a distinct mechanism from the existing four mechanisms for this reaction. The details of this mechanism will be discussed. 

Edmunds Z. Reineks, Xuemei Zhang, and ANTHONY J. BERDIS 

Department of Pharmacology and the Ireland Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106 

The most crucial step in mutagenesis arguably occurs during DNA replication and is facilitated by the ability of the DNA polymerase to insert dNTPs opposite and beyond unrepaired DNA lesions. An attractive approach to combat mutagenesis is through the administration of novel nucleoside analogs that are selectively inserted opposite damaged DNA but cannot be extended once inserted, i.e., chain terminators. We have employed several principles of rational drug design to synthesize nucleoside analogs that could effectively compete for the misinsertion of natural dNTPs opposite an abasic site, a prototypical non-coding DNA lesion devoid of hydrogen bonding potential. Of a panel of analogs tested, 5-nitro-1-indolyl-2'-deoxyribose-5'- triphosphate (5-NITP) proved to be unique since it is efficiently inserted opposite an abasic site. The enzymatic insertion of 5-NITP is 1,000-fold more efficient than that measured for natural dNTP insertion opposite an abasic site, thus providing selectivity. This higher efficiency results from a substantially increase in the rate of catalysis rather than in binding affinity. Replacement of the nitro group with -H or -F reduces the rate of catalysis by -1,000-fold and emphasizes the role of л-electrons in enhancing catalysis. Equally important is the fact that the polymerase is unable to continue elongation from the damaged primer/template thus proving that 5-NITP acts a potent chain- terminator during translesion DNA replication. The results of these experiments are discussed within the context of employing 5-NITP as a chemotherapeutic agent to prevent translesion DNA replication and/or as a diagnostic tool for the detection of DNA damage. 

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