Program

Todd Weaver and Leonard Banaszak

Department of Biochemistry, University of Minnesota, Minneapolis, MN 55405 

Fumarase catalyzes the interconversion of L-malate and fumarate as part of the citric acid cycle. Fumarase C from E. coli is homologous with the non-metal containing fumarases in eukaryotic cells as well as aspartase, adenylo-succinate and arginino-succinate lyase, and 8-crystallin. The crystal structures of native fumarase C along with inhibited forms have been determined and refined to about 2 Å resolution. The x-ray structures show that the fumarase C tetramer is formed by a core of 20 a-helices - 5 coming from the central domain of each subunit. In addition to the central 5 helical core, there are two other structural domains. The active site occurs in a crevice formed at the interface of three of the four subunits. However, a second nearby site has been identified in the electron density maps. The second site which could be some sort of activation or regulatory site is approximately 12 Å from the active site. The active site has been analyzed in terms of potential catalytic sidechains in both the citrate and pyromellitic acid inhibited forms. Sidechains of a histidine, glutamic acid, lysine, and asparagine along with a water molecule have been identified at the active site. Their potential roles in a catalytic cycle will be discussed. 

Gregory K. Farber 

Department of Biochemistry and Molecular Biology The Pennsylvania State University 108 Althouse Laboratory University Park, PA 16802 

In recent years, there has been a great deal of interest in the behavior of enzymes in nonaqueous solvents. Enzymes often exhibit increased thermostability or altered substrate specificity in organic solvents, and in these solvents enzymes are able to catalyze reactions that are either kinetically or thermodynamically impossible in water. A final advantage of exploring nonaqueous enzymology is that it is possible to trap enzyme substrate complexes in organic solvents that have only a very short lifetime in aqueous solutions. 

We have solved a number of crystal structures in organic solvents to try and explain the altered properties which have been observed. The structure of gamma chymotrypsin in a solution of hexane and isopropanol has suggested an explanation for altered substrate specificity. A series of structures of subtilisin in various concentrations of dimethylformamide has suggested the explanation for the lower kcat which is often a consequence of moving into an organic solvent. Finally, several different solvents have been used to trap all of the important intermediates in the reaction catalyzed by chymotrypsin. 

Xuejun Zhong, Smita S. Patel, Brian G. Werneburg, and Ming- Daw Tsai

Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210 

Two conformational changes induced consecutively by nucleotide binding and catalytic Mg2+ ion binding have been identified in the mechanism of rat DNA polymerase B. Correct nucleotide binding efficiently induce a fast initial conformational change which allows catalytic Mg2+ ion to bind to the enzyme, DNA and dNTP in the catalytic site at the same time and then further induce the second rate-limiting conformational change before chemistry. While incorrect nucleotide can only induce the initial conformational change at about 50 fold higher concentration with similar rates (control fidelity through productive binding) and undergoes the second conformation at about 500 fold slower (selection through productive catalysis). We suggest that the dNTP binding step can be divided into a nonspecific binding step involving only the ẞ,y phosphate and an equilibrium step of "base pairing". Selection through productive binding is achieved by allowing only "base-paired" population can induce the initial conformation change. Initial conformational change induced by incorrect nucleotide binding results in a "bad fit" and greatly raises the kinetic energy barrier of the second conformational change step (the rate limiting step) which controls fidelity through productive catalysis.

John J. Hlavaty and Thomas Nowak* 

Department of Chemistry and Biochemistry University of Notre Dame, IN 46556 

Avian liver mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) is a 67 kDa monomeric gluconeogenic enzyme that catalyzes the reversible GTP dependent conversion of OAA to PEP, GDP and CO2. PEPCK shows an absolute requirement for divalent cations for activity and Mn" is the best activator. Mixed metal kinetic studies show a dual cation role for PEPCK. One cation activates the enzyme through a direct interaction with the protein at site n. The second cation acts in the cation-nucleotide complex that serves as a substrate at site n2. Recently, an active Co3*(n,)-PEPCK_complex has been formed. Co3*(n)-PEPCK provides an excellent tool for examining the kinetic, mechanistic and binding properties of the n2 metal without the concern of metal binding at the n, site. EPR studies performed on the Co3*(n)-PEPCK-GTP complex have determined a stoichiometry of one mole Mn2+ bound per mole Co3*(n,)-PEPCK-GTP with a KD = 5 μM. Water proton relaxation rate (PRR) studies show a significant enhancement for the Co3*(n1)-PEPCK-GDP-Mn2*(n2) complex in the presence of PEP but not with OAA or CO2 suggesting that PEP interacts with the second metal ion. A PRR study for both the Co3*(n,)- PEPCK-GTP-Mn2*(n2) and Co3*(n,)-PEPCK-GDP-Mn2 (n2) complexes as a function of frequency (1) estimated the hydration number of the n2 metal at a value between 0.5 to 0.7. A temperature dependence of the rates of relaxation determined that the water protons at the second metal site are in fast exchange with an activation energy of -1.97 kcal/mol for the Co**(n)- PEPCK-GTP-Mn2*(n2) complex. The metal-metal distance between the enzyme-bound Mn2* at site n, and Cr3*-GTP at site n2 as determined by PRR techniques is 8.5 Å. To determine the location of the n2 cation site on PEPCK, the Co3*(n)-PEPCK complex was incubated with Co2*, GTP and H2O2 creating a doubly labeled and inactive Co3*(n,)-PEPCK-GTP-Co3*(n2) complex. The Co3*(n1)-PEPCK-GTP-Co3*(n2) complex was digested by LysC and two cobalt-containing peptides were purified using reverse phase HPLC. The first cobalt-containing peptide has previously been identified as the n, site. Amino acid sequencing on the second cobalt-containing peptide identified the region of Tyr57-Lys76 of PEPCK. This is a highly conserved region located by the N-terminus of PEPCK near the putative PEP binding region. Capillary electrophoresis analyses of tryptic and chymotryptic digests of the second cobalt-containing peptide suggest that Asp69 and Glu74 may serve as ligands to the site n2 metal. 

Research supported by NIH grant DK17049 and the GAANNP Fellowship, Project Award Number P200A20261. 

Vahe Bandarian & George H. Reed

Institute for Enzyme Research, University of Wisconsin-Madison, Madison WI 53705. 

Ethanolamine ammonia lyase (EAL) catalyzes the deamination of ethanolamine to form acetaldehyde and ammonia - a transformation that requires coenzyme B12. The function of coenzyme B12 is to abstract a hydrogen atom from C1 of the substrate, ethanolamine, to generate a free radical intermediate. This intermediate then rearranges to form a product radical which reaquires the hydrogen that had been abstracted from C1. Deamination of the carbinolamine affords acetaldehyde and ammonia. The presence of free radicals during catalysis by EAL was shown in a seminal paper by Babior and collaborators [Babior, B.M., Moss, T.H., Orme-Johnson, W.H., Beinert, H. J. Biol. Chem. 249, 4537-4544] in which the authors showed that upon incubation of propanolamine, a slow substrate, with coenzyme B12 and EAL, an EPR signal was observed which is consistent with formation of a substrate-based free radical. This signal was called the "radical doublet" signal and similar signals have been observed in a number of other coenzyme B12 enzyme systems. While these EPR signals were observed over 20 years ago, the exact identity of the species giving rise to the EPR signals have remained unsolved mysteries. The identity of the free radical giving the “radical doublet" EPR signal observed with EAL will be the focus of the presentation. The kinetic competence of the signal has been established by rapid freeze- quench EPR. A large number of isotopically labeled substrate analogs have been synthesized and incubated with EAL and with coenzyme B12 and studied by EPR. The spectra have been extensively analyzed and the simulation parameters identify the intermediate. The structure of the intermediate foreshadows the steps that are to come in the transformation of the substrate to products. Furthermore, the structure of the radical emerging from the EPR studies suggest how an enzyme deals with unstable species in its active site. This presentation will show the progress made towards assignment of the "radical doublet" signal observed with EAL.

Alexey Bulychev, Irina Massova, and Shahriar Mobashery*

Wayne State University, Department of Chemistry, Detroit, MI 48202-3489. 

The ß-lactamase activity is the primary means for bacterial resistance to ß-lactam antibiotics. These enzymes are presumed to have evolved from the primordial cell-wall biosynthetic enzymes, the modern forms of which are referred to as penicillin-binding proteins (PBPs). Class A ß-lactamases are most common among pathogens, whereas class C enzymes are second most common. Both classes A and C of B-lactamases, as well as PBPs, undergo acylation at an active-site serine residue by ß-lactam antibiotics. The rate of deacylation of this acyl-enzyme intermediate from the active site of penicillin-binding proteins is slow, thereby the bacterium is deprived of the biosynthetic function of these enzymes, an events that results in bacterial death. However, ß-lactamases are capable of undergoing deacylation in a facile manner, completing the turnover necessary for hydrolysis of the B-lactam antibiotic. We have investigated the mechanistic details of the deacylation step in both classes A and C of B- lactamases. A molecular probe, 6α-hydoxymethypenicillanate (1), was designed in a computer- aided process with the help of the crystal structure for the Escherichia coli TEM-1 ß-lactamase, a prototypic class A enzyme. This molecule was designed to prevent the approach of the presumed hydrolytic water from the o-face of the acyl-enzyme intermediate. The compound was synthesized and studied with the purified enzyme. The compound acylated the enzyme readily, but resists deacylation, as expected. The crystal structure for the acyl-enzyme intermediate, the first for any acyl-enzyme intermediate for turnover of a substrate by a native class A ß-lactamase, supported the design paradigms, indicating that the approach of the hydrolytic water is from the a-face, and is promoted by Glu-166 as a general base. Interestingly, there is no counterpart to Glu-166 in class C B-lactamases. Earlier work from our laboratory with the Enterobacter cloacae P99 B-lactamase, a prototypic class C B-lactamase had suggested that the approach of the hydrolytic water may be from the B-face of the acyl-enzyme intermediate in this case (J. Am. Chem. Soc. 1995, 117, 4797). If this were the case, the formerly ß-lactam nitrogen, now a secondary amine at the acyl-enzyme intermediate stage appeared to us ideally positioned to serve as the general base in promoting a water molecule for approach to the acyl carbonyl from the B- face. To test this possibility two molecules were synthesized, p-nitrophenol (2R,5R)-5- prolylacetate (2) and p-nitrophenol (15,3S)-3-carboxy-cyclopentylacetate (3). Compound 2 acylates the active site serine of the P99 enzyme, and the intermediate undergoes deacylation. On the other hand, compound 3 only acylates the active site, and not having the requisite amine in its structure, the intermediate resists deacylation. Both compounds serve as substrates for the class A TEM-1 B-lactamase, as was expected. We conclude that substrate-assisted catalysis applies for the class C ß-lactamases. On the basis of the evidence discussed here, the knowledge of the crystal structures for the TEM-1 and P99 B-lactamases and the Steptomyces R61 DD- peptidase/transpeptidase (a PBP) and other mechanistic features of these proteins, we propose that class C B-lactamases should be more ancient than class A enzymes.

C.A. Sheppard, P. Goyette+, P. Frosst+, R. Rozen+, and R.G. Matthews" 

*Department of Biological Chemistry, Cellular and Molecular Biology and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109

+Department of Pediatrics, Human Genetics, and Biology, McGill University, Montréal, Quebec 

Deficiencies in methylenetetrahydrofolate reductase (MTHFR) have been correlated with increased risk of heart disease and neural tube defects. Human MTHFR has been cloned and mutations identified in human patients with altered MTHFR activity. A human MTHFR alanine to valine mutation was identified as a genetic risk factor for cardiovascular disease and neural tube defects. Noting that the E. coli MTHFR (ecMTHFR) shows significant homology to human MTHFR, and that mutations causing altered function in humans occur in regions of high identity, we constructed the corresponding changes in ecMTHFR. Mutations associated with loss of enzymatic activity in humans caused loss of activity in E. coli; thus, ecMTHFR is a useful model for studying the MTHFR family. Purified Ala177Val protein has catalytic properties similar to wildtype ecMTHFR, but has impaired flavin binding. The rate of flavin and activity loss is reduced by the addition of folate. These results suggest that in humans with this mutation, impaired flavin binding results in decreased amounts of active MTHFR enzyme. Further studies are in progress using the histidine tagged wildtype and Ala177Val enzyme. In addition, collaboration with Dr. Brian Gunther and Dr. Martha Ludwig has reproducibly generated crystals of ecMTHFR that diffract to 3.2Å.

James A. Peliska*, Sam Gabarra*, Lynn Hupe#, Donald Hupe#,* 

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

#Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105

Over the last several decades, extensive research on the infection cycle of the retrovirus has revealed important details concerning its structure, molecular biology, enzymology and pathogenicity. An important milestone in the study of retroviruses came in 1970 with the discovery and isolation of viral RNA-dependent DNA polymerase (reverse transcriptase) by Temin and Baltimore. Interest in retrovirology intensified in the mid 1980s with the discovery of the virus HIV as the required agent for acquired immunodeficiency syndrome (AIDS). A major emphasis of this research has been on developing anti-retroviral drugs that would affect particular stages of retroviral replication, with reverse transcriptase as a principle target. A major complication in the development of a therapy for HIV is the high degree of genetic variation associated with its RNA genome. These variations result primarily from the low fidelity associated with proviral DNA synthesis by reverse transcriptase, and the high frequency of genetic recombination occurring during reverse transcription. This results in the spread of a broad population of genetically distinct HIV virion and the rapid selection of drug resistant viral strains. Our laboratory is developing strategies to elucidate the mechanistic details of one class of recombination events- termed forced copy-choice- that occurs during reverse transcription. Since these DNA strand transfer events occur frequently during reverse transcription, they make attractive new targets for therapeutic intervention. Such inhibitors could both inhibit viral replication directly, and serve to curtail the level of genetic recombination that occurs during reverse transcription thereby helping to stabilize the viral genome. Using a DNA strand transfer model system and a technique called scintillation proximity, we have applied drug screening technology developed in this laboratory to the discovery of new inhibitors of HIV-1 reverse transcriptase. The screen was specifically designed to identify inhibitors that target DNA strand transfer events catalyzed by the target enzyme during reverse transcription and recombination. Distinct classes of novel HIV-1 RT inhibitors have been identified using these technologies. The mechanism by which these new inhibitors function to inhibit recombination is under investigation using biophysical and enzyme kinetic techniques. 

Mengmeng Fu and Richard B. Silverman 

Department of Chemistry; Northwestern University; Evanston, IL 60208 

4-Amino-4,5-dihydro-2-thiophene carboxylic acid (1, X = S) and 4-amino-4,5- dihydro-2-furan carboxylic acid (2, X = O) are rationally designed mechanism-based anticonvulsant agents[1],[2] based upon the known mechanism of the dihydroaromatic y- aminobutyric acid aminotransferase (GABA-AT) inhibitors gabaculine and isogabaculine (Scheme 1, aromatization pathway).B] It was believed that 1 and 2, when incubated with GABA-AT, would be converted to aromatic pyridoxamine-5'-phosphate adducts. Our study of R-1, S-1 and S-2, however, has shown that all of these compounds inactivate pig brain GABA-AT via mechanisms different from the proposed aromatization pathway. Despite the fact that GABA-AT has been shown to abstract only the 4-pro-S-hydrogen from the substrate GABA, HPLC and mass spectrometric studies of metabolites formed have shown that both R-1 and S-1 inactivate GABA-AT by the same mechanism (Scheme 2, enamine pathway), with R-1, significantly slower, as expected. The mechanism of S-2 inactivation of GABA-AT is expected to be different from that of 1, because oxygen is not as good a leaving group as is sulfur. The metabolites formed from S-2 inactivation of [3H]-pyridoxal-5'-phosphate-GABA-AT coeluted with cold pyridoxamine-5'-phosphate (PMP). The mass spectrum, however, showed the presence of PMP and an unknown species with a mass 22 units greater than PMP. This suggests the possible involvement of a Michael addition pathway for S-2. 

[1] Burkhart, J. P.; Holbert, G. W.; Metcalf, B. W. Tetrahedron lett. 1984, 25, 5267.

[2] Adams, J. L.; Chen, T. M.; Metcalf, B. W. J. Org. Chem. 1985, 50, 2730. 

[3] Nanvati, S. M.; Silverman, R. B. J. Med. Chem. 1989, 32, 2413-2421.