Program

EVELYN JABRI, Peter Mikulecky, Paras Ramolia, and Eric Espinosa

Indiana University, Department of Chemistry, Bloomington, IN 47405. 

The GIR1 family of introns is the single example of group I introns with a biological function other than splicing. These <200 nt RNAs have evolved to be efficient autolytic RNAs, performing site-specific hydrolysis at a rate two orders of magnitude faster than that of the 3′SS cleavage of canonical group I introns. This evolution has resulted in an unusual structural organization, including a pseudoknot (P15) within the catalytic core [1]. An in vitro selection experiment identified RNAs that perform site-specific hydrolysis at a faster rate [2]. We are using these data to understand how various nucleotides in GIR1 contribute to rate enhancement. Overall, the results show that variants with dramatically improved hydrolytic activity have only 4-12 mutations relative to the wild type RNA. Three mutations within or proximal to P15 act synergistically to increase activity 50-fold. The current hypothesis is that P10 is docked in the ground state of the selected RNAs and undocked (or docked less efficiently) in wild type GIR1. Specifically, two mutations change the conformation of P15, which in turn allows the movement of P10 into a catalytically more favorable position. Additional mutations within P10, increase the efficiency and specificity of its interactions with the core, resulting in additional rate enhancement. This hypothesis is being tested by using various structure probing methods including X-ray crystallography, nucleotide analogue modification interference and hydroxyl radical footprinting on both the wild type and selected variants of GIR1. Progress towards identifying the role of specific nucleotides in enhancing the catalytic rate of GIR1 will be presented. 

1. Jabri, E., S. Eigner, and T. Cech, Kinetic and secondary structure analysis of Naegleria andersoni GIRI, a group I ribozme whose putative biological function is site-specific hydrolysis. Biochemistry, 1997. 36(51): 16345-54. 

2. Jabri, E. and T. Cech, In vitro selection of the Naegleria GIR1 ribozyme identifies three base changes that dramatically improve activity. RNA, 1998. 4(12): 1481-92. 

Shu-ou Shan, Aiichiro Yoshida, Alex Kravchuk, Jimmy Hougland, Dan Herschlag,, JOSEPH PICCIRILLI

University of Chicago, Departments of Biochemistry & Molecular Biology and Chemistry, Chicago, IL 60637. 

Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, experiments with thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with 3'- and 2'- substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the non-bridging pro-S, oxygen of the reactive phosphate group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-Sp oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-Sp oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion interactions with substrate for any RNA or protein enzyme catalyzing phosphoryl transfer.

JOAN B. BRODERICK, Wei Hong, William E. Broderick, Timothy F. Henshaw, Jennifer Cheek, Danilo Ortillo, Carsten Krebs, Boi-Hanh Huynh, Charles Walsby, and Brian M. Hoffman 

Pyruvate formate-lyase activating enzyme (PFL-AE), which generates the catalytically essential glycyl radical on PFL, is a representative member of an emerging group of enzymes that utilize iron-sulfur clusters and S- adenosylmethionine (AdoMet) as required cofactors in radical generation. This group includes related activating enzymes such as the anaerobic ribonucleotide reductase activating enzyme from E. coli, as well as biotin synthase, lipoic acid synthase, and lysine aminomutase. Though diverse in function, these enzymes have been proposed to have in common key mechanistic features including the generation of an intermediate 5'- deoxyadenosyl radical that initiates catalysis by hydrogen atom abstraction. This presentation will focus on our recent work in characterizing the iron-sulfur cluster of PFL-AE and its role in generation of the putative 5'-deoxyadenosyl radical intermediate. We have shown that PFL-AE, which contains primarily [3Fe-4S]* clusters upon anaerobic isolation, is reductively converted to the [4Fe-4S] form upon reduction. The [4Fe-4S]* state of PFL-AE has been shown to be catalytically active, donating the electron required for reductive cleavage of AdoMet and subsequent generation of the glycyl radical of PFL. Finally, we will provide evidence that AdoMet is closely associated with the [4Fe-4S] cluster of PFL-AE, suggesting that the cluster is directly involved in the radical generation chemistry.

BRIAN G. FOX 

University of Wisconsin Department of Biochemistry 1710 University Avenue Madison WI 53705 

Stearoyl-ACP delta-9 desaturase (delta-9D) catalyzes the NADPH and O2 dependent, regio- and stereo-specific insertion of a cis double bond at the C9 position of 18:0-ACP to produce 18:1-ACP. delta-9D is a member of the soluble diiron enzyme family, which also includes ribonucleotide reductase, bacterial hydrocarbon hydroxylases, and ferritins. The importance of binding interactions between the complex substrate (acyl chain and protein, 8 kDa) and delta-9D to the catalytic cycle will be discussed. Rapid mix of the aerobic complex of 18:0-ACP and delta-9D with reduced ferredoxin gives a pseudo first order exponential burst of product formation with amplitude corresponding to the turnover of both active site of the homodimer. In the presence of excess ferredoxin, the burst phase was followed by a continued linear phase of product formation whose rate matched Kcat for the steady-state appearance of 18:1-ACP. Use of stopped-flow fluorescence anisotropy revealed equilibrium dissociation constants and dissociation rate constants for dansyl- and fluoresceinyl-labeled acyl-ACPs with resting and chemically 4e reduced delta-9D. The results show that Koff is most strongly influenced by acyl chain length, and thus provides an important kinetic input to the observed chain length selectivity. Single turnover desaturation in the presence of 1802 and the reaction of substrate analogs revealed unique patterns for reactivity as compared to other soluble diiron enzymes and to the broader class of integral membrane fatty acid desaturases. The combination of catalytic and structural results are used to suggest a minimal mechanism for reaction of the soluble desaturase complex.

S. RAMASWAMY1, Andreas Karlsson3, Zulfiquar Malik1, Juanito V.Parales1, Rebecca E. Parales1, Michael Larkin2, David T. Gibson1 and Hans Eklund3. 

1Department of Biochemistry and Microbiology, University of Iowa, Questor Center, Queens University of Belfast and 3Swedish University of Agricultural Science, Uppsala, Sweden. 

Naphthalene-1,2-dioxygenase (NDO) belongs to the large class of Rieske non-heme iron oxygenases (ring hydroxylating dioxygenases) including biphenyl dioxygenase, phthalate dioxygenase, etc. 

These enzymes catalyze the cis-dihydroxylation of a variety of two and three ring aromatic compounds. NDO catalyzes the conversion of naphthalene to cis- (1R,2S)dihydroxy-1,2-dihydronaphthalene. The reaction consumes two electrons and a dioxygen molecule. In addition to ring dihyroxylation, NDO also catalyzes a variety of other reactions including, mono- hydroxylation, desaturation, O and N-dealkylation and sulfoxidation. In order to understand the reasons for regio-and stereo specificity of this class of dioxygenases, several mutants have been made with modified stereo-and regio specificities. The structures of several complexes of the protein (with substrates/products/ intermediates/ NO) provide insight into the mechanism of action. While the catalytic iron in the active site of NDO from the Psuedomonas is tetrahedrally coordinated the mono-iron in the NDO from Rhodococcus shows a distorted bipyramidal co-ordination. Comparison of the active site of the two structures is now in progress and the results will be presented. 

HARRISON, DAVID H. T.(1), Marks, Greg M.(1), Harris, T.K.(2), Messiah, M.A.(2), Mildvan, A.S.(2); 

1-Medical College of Wisconsin, 2-The Johns Hopkins Medical School 

Methylglyoxal synthase (MGS) catalyzes the elimination of phosphate from DHAP to form the enol of methylglyoxal, which then tautomerizes to methylglyoxal. Although the three dimensional folds of MGS and triosephosphate isomerase (TIM) are different, the substrate, the enzyme- bound enediolic intermediate, and the positioning of the active site carboxylate and imidazole are the same. This suggests that the chemical mechanisms are also likely to be the same. However, inhibitor binding studies suggest that the transfer of protons from one oxygen to another may proceed by a non-'TIM-like' mechanism. Phosphoglycolohydroxamic acid (PGH) is an analogue of the enediolic intermediate that binds to both MGS and TIM with a K of 40 nM and 4 μM, respectively, whereas phosphoglycolic acid (PGA) binds to both MGS and TIM with a K¡ of 4 μM. Further, the MGS-PGH complex structure shows a short strong hydrogen bond between the NOH of PGH and the carboxylate of Asp 71 of MGS. A novel mechanism is presented that explains this data and is consistent with the "Woodward and Hoffman" rules. 

KENNETH W. OLSEN, Stefan Fischer, Flor Torres, Timika Hoffman-Zoller, Amanda Jonson, and Martin Karplus

Loyola University Department of Chemistry 6535 N. Sheridan Rd. Chicago, IL 

A structurally continuous path for the allosteric transition of hemoglobin has been calculated with the Conjugate Peak Refinement method (Fischer & Karplus, Chem. Phys. Let. (1992) 194:252). The result is a minimum energy path obtained without any constraint to drive the reaction, giving an energetically plausible sequence of events linking the T- and R-states in atomic detail. The important salt bridges break early, before the quaternary "switch" occurs synchronously in both a1/B2 interfaces. However, the simulation reveals important aspects of the transition that cannot be predicted from the end-states. Changes in the two aẞ dimers occur nearly synchronously, conserving an overall C2 symmetry during the transition. The quaternary change occurs in the a-chains significantly before it occurs in the B-chains, defining two main quaternary events. Rotation around the G-helix of each a-chain in the first quaternary transition brings the aH-helices into a position so that they can serve then as hinges for the subsequent "switch". The present simulation shows which elements of the secondary structure actually serve as pivoting axes. The two main quaternary events are separated by a transient state, whose central cavity between the a-chains is small as in the R-state, whereas the B-chains are more T-like. The tetrameric nature of the molecule is required for this transition, but there are also changes in the conformations of the individual subunits. Continuous paths for the structural changes in the isolated subunits were calculated. Both isolated subunits can accomplish approximately 50% of their conformational change before the first saddle point, which is significantly different than the path for the entire tetramer, demonstrating the quaternary constraints placed on each of the subunits. 

PETER A. TIPTON

Dept. of Biochemistry, University of Missouri, 117 Schweitzer Hall, Columbia, MO 65211. 

Urate oxidase catalyzes the dioxygen-dependent oxidation of urate, a reaction that is of significant metabolic importance in leguminous plants, most mammals, and some microorganisms. Although the product of the reaction has been widely considered to be allantoin, recent studies have shown that it is 5-hydroxyisourate, a metastable species that decays to allantoin. Mechanistic interest in the catalytic reaction derives largely from the observation that the enzyme neither contains nor requires a transition metal or organic cofactor. We have proposed that the enzyme is able to catalyze the direct reaction between dioxygen and urate to form urate hydroperoxide as a key intermediate in the catalytic cycle. Two intermediates have been detected by stopped-flow spectroscopic studies. One is proposed to be urate hydroperoxide; the other is believed to be the dianion of urate. Ab initio calculations reveal that oxidation of the urate dianion is highly favorable. 180 labeling studies have demonstrated that the urate hydroperoxide intermediate can be reduced at the active site by cysteine or dithiothreitol. The observation that urate oxidase generates 5- hydroxyisourate instead of allantoin begs the question of the mechanism of allantoin biogenesis. We have identified a novel enzyme in soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is also the first step in the nonenzymatic pathway to allantoin formation. We have cloned the gene encoding the enzyme, which we designate hydroxyisourate hydrolase.

Ji Hyun Lee, Kathy Z. Chang, Vishal Patel, and CONSTANCE J. JEFFERY

University of Illinois at Chicago, 1899 Maple Avenue, Hanover Park, Illinois 60133 

Phosphoglucose isomerase (PGI; E.C. 5.3.1.9) catalyzes the second step in glycolysis, the interconversion of glucose 6-phosphate and fructose 6- phosphate. PGI also has roles in gluconeogenesis and the glycosylation of proteins. The catalytic mechanism involves acid-base catalysis with a cis- enediol(ate) intermediate. Through a series of crystal structures, solved at between 1.8 and 2.1Å resolution, we have developed a model for the roles of active site amino acid residues, ordered water molecules, and conformational changes in the multistep catalytic mechanism. The crystal structure of a complex with cyclic fructose 6-phosphate bound in the active site indicates roles for His388, Lys518, and an ordered water molecule in the ring opening step. Complexes with inhibitors that mimic the cis-enediol (ate) intermediate of the isomerization step identify roles for Glu357, Arg272, and additional ordered water molecules in the isomerization step.