1985 — 1988 |
Kozarich, John W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Enzyme Mechanisms in Three Carbon Metabolism @ University of Maryland College Pk Campus
This grant seeks continued support for our research on the mechanism of enzyme action. Specifically, we propose to investigate three enzymes which are involved in the metabolism of three-carbon units. Our work on glyoxalase I will focus on the identification of the active site base responsible for proton transfer using some of the principles of our recent finding of "mirror-image" catalysis by glyoxalase I. In addition, other studies are designed to probe the nature of the partitioning reaction of halomethylglyoxals by the enzyme. We will attempt to determine the contributions of product release from the enzyme and halogen orientation on the partitioning reaction. Our studies on lactate racemse will focus on two major issues. First, we will investigate the question of proton versus hydride transfer in the racemization step. We will accomplish this using many of the techniques that we have established in the glyoxalase I studies. Second, we will address the question of substrate activation in this system. There has been some speculation that lactate racemase may involve a covalent enzyme-substrate complex or a "charged" enzyme. Extreme difficulty in the purification and stabilization of this enzyme has prevented the resolution of this issue. We propose to develop a general method for the detection of a "charged" enzyme which we call anhydride detection by intermolecular oxygen scrambling. The technique relies on the implied radomization of carboxylate oxygens in an acyl exchange mechanism. Our technique will involve a novel utilization of 18O perturbations on 13C NMR resonances. This method will be extended to other enzymes where "charged" forms and anhydride intermediates have been proposed. Finally, we would like to initiate studies on elucidating the molecular mechanism of pyruvate-formate lyase, an enzyme which bears some similarity to the two discussed above in terms of ambiguity of electron flow. We propose studies designed to determine the direction of bond cleavage, the intermediates formed on the enzyme, and the function of enzyme activation by SAM, flavodoxin, and Fe(II)-protein complex.
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0.988 |
1985 — 1992 |
Kozarich, John W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Drug-Induced Dna Degradation @ University of Maryland College Pk Campus
This proposal seeks continued support for our studies directed toward the elucidation of the molecular mechanisms of DNA degradation by a variety of metal complexes, in particular the metallobleomycins (BLM). During the next funding period, we plan a comprehensive program which will include the following studies: 1. A complete analysis of the kinetic isotope effects on 4'- hydrogen abstraction by BLMs. We have designed methods to permit an accurate determination of these effects at individual sequence sites in a defined pBR322 fragment. This will allow us to study the potential effects of local conformation on the chemistry of the DNA-BLM interaction. 2. Continued investigations on the mechanism of deoxyribose ring fragmentation by Fe.BLM using 180 analysis. Our recent work has established that 180 incorporation into the phosphogycolate moiety is derived from the second 02 required for base propenal formation. Further studies are planned that will discriminate among several proposed pathways leading to cleavage. 3. The chemistry of BLM action on other DNA forms. Evidence suggests that BLM can react with A-form DNA. Model analysis has revealed that the chemistry of this process maydiffer from B DNA due to an altered accessibility of sugar hydrogens. Using techniques which we have developed, we will determine the mechanism of cleavage in appropriate model DNA's and DNA- RNA hybrids. Z DNA will also be studied. 4. The chemistry of other metallobleomycins. We plan to investigate the cleavage of DNA by cobalt-BLM and manganese- BLM. These complexes exhibit different activational requirements than the iron complex and yield different products. 5. The mode of BLM binding to DNA. The question of an intercalative vs. nonintercalative process for BLM binding remains open. Studies are planned to distinguish between these possibilities. 6. Model studies with other DNA cleaving agents. The probes which we have developed should be valuable in the determination of the mechanistic features of other agents which cleave DNA by hydrogen abstraction mechanisms. Ruthenium and cobalt complexes of 4,7-diphenylphenanthroline, methidium propyl- EDTA-Fe(II), and the 1,10-phenanthroline-copper complex are recent literature examples which are amenable to analysis by our technology.
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0.988 |
1985 |
Kozarich, John W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanistic Approaches to Drug Design @ University of Maryland College Pk Campus
This proposal seeks support for the development of strategies of drug designs which are based on the novel mechanistic properties of enzymes postulated to be the targets in cancer chemotherapy and in the treatment of other diseases. Specifically, during the initial tenure of this grant, we propose to develop a new class of potential mechanism-based inhibitors of glyoxalase I which are "inverse substrates" for this enzyme and rely on our recently discovered mechanistic observations, termed mirror-image catalysis, for their activation to reactive species. We will use some of the assumptions implicit in mirror-image catalysis as a probe for determining the absolute stereochemistry of the glyoxalase I reaction, a question which has eluded solution due to the rapid equilibration of the diastereomeric thiohemiacetals in the normal reaction. The intrinsic stability which may be incorporated into the inverse substrates will permit stereochemical analysis by a number of classical methods. Secondly, the principles of mirror-image catalysis will permit the synthesis of stable, glutathione-containing inverse substrates which may undergo conversion to highly reactive compounds capable of inactivating the enzyme and/or exhibiting cytotoxicity in an in vivo situation at some other site. These compounds will be synthesized by techniques which have been developed in this laboratory. Their mechanistic and kinetic properties will be thoroughly studied with purified glyoxalase I from a number of sources and inhibitory activity will be determined in cell culture. The function of the glyoxalase system has been a point of controversy for nearly seventy years. It has been purported to be merely a detoxification pathway by some researchers to a basic regulatory system of cell growth by others. These studies should shed light on its function and have applicability to the study of other glutathione-dependent processes, such as inflammation and allergic responses.
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0.988 |
1986 — 1993 |
Kozarich, John W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Enzymes of Aromatic Acid Metabolism @ University of Maryland College Pk Campus
Our long term goals in this research is to thoroughly investigate the rich mechanistic and stereochemical questions associated with the enzymes of aromatic acid matabolism. These enzymes, particularly those constituting the Beta-ketoadipate pathway, are of considerable interest from an intellectual as well as practical standpoint. These studies should shed light on the role of mechanism and stereochemistry in the evolution of these enzymes as they adapted to specific functions and specific substrates. In addition, the biodegradation of aromatics is becoming increasingly important as a potential solution to the problem of polution control. Our specific aims during the funding period will be: (1) A study of the mechanism and stereochemistry of the enzymes of the Beta-ketoadipate pathway from P. putida. (2) The analysis of the reactions of haloprotocatechuates with the P. putida enzymes. (3) A study of the mechanism and stereochemistry of enzymes of A. calcoaceticus, particulary the two enol-lactone hydrolases. (4) An analysis of the Pseudomonas sp B13 enzymes, particularly the specific enzymes responsible halocatechol metabolism. (5) A study of the enzymes from the eukaryote, Trichosporon cutaneum, which appear to have different specificities. We believe that these basic studies should serve as a foundation for the genetic manipulation of microorganisms to meet specific detoxification needs. Furthermore, the studies will increase our mechanistic understanding of these intriguing enzymes.
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0.988 |
1989 — 1993 |
Kozarich, John W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Enzyme Action @ University of Maryland College Pk Campus
This competitive renewal application seeks continued support for our major research effort directed toward the elucidation of fundamental new chemistry of enzyme and inhibitor action. During the next funding period, we will continue our work in three major areas: studies on the activation and chemical mechanism of E. coli pyruvate formate-lyase, further investigations on the mechanism of halide eliminations through intervening aromatic rings by enzymes which catalyze carbanionic mechanisms and some final studies on the stereospecificity of glyoxalase I catalysis. Our effort will focus on the key enzyme of prokaryotic anaerobic glycolysis, pyruvate formate-lyase (PFL). Recent work, including our own, has provided suggestive evidence that the reaction may proceed by an enzymatically unprecedented homolytic cleavage of pyruvate. Our approach will involve a thorough study of the mode inactivation of PFL by hypophosphite, acetylphosphinate and propargylic acid. The latter two were recently discovered by us to be extremely potent inactivators of the enzyme. The novel mode of activation of PFL will also be investigated. These studies will require a broad effort with expertise in synthesis, enzyme purification, stable and radio isotope analyses, high field NMR and EPR studies. We have recently demonstrated that novel quinodimethane intermediates are generated from p-halomethyl-substituted aromatic substrates by enzymes which proceed by putative carbanionic mechanisms. The phenomenon has been established for glyoxalase I, benzoyl-formate decarboxylase and mandelate racemase. These studies will now be extended to the flavin-dependent D- amino acid oxidase and, in an attempt to determine if radical mechanisms also can catalyze these eliminations, to monoamine oxidase. This work is of particular significance to the area of drug design and enzyme inactivation by quinone methide intermediates. Finally, we will execute some final experiments to definitely establish that glyoxalase I has the unique and evolutionarily significant ability to catalyze a non-stereo-specific proton abstraction from one carbon followed by a completely stereospecific protonation at an adjacent carbon by single active site base. We believe that these studies are relevant to a number of basic issues in biochemistry and medicine and that new chemistry and enzymology will result from this work.
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0.988 |
1993 — 1995 |
Gerlt, John Kozarich, John Armstrong, Richard Mariano, Patrick (co-PI) [⬀] Dunaway-Mariano, Debra (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of a Computer Workstation @ University of Maryland College Park
These investigators will purchase a computer workstation, peripheral equipment, and software. The equipment will be used for modeling studies and molecular dynamics calculations applied to proteins and nucleic acids, for DNA sequence searches, and for molecular mechanics calculations on small molecules. Enzymes to be studied include glutathione S-transferase, epoxide hydrolase, 4- chlorobenzoate dehalogenase, staphylococcal nuclease, and mandelate racemase.
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0.915 |
1993 — 1995 |
Kozarich, John Poli, Rinaldo [⬀] Eichhorn, Bryan (co-PI) [⬀] Falvey, Daniel (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Upgrade of An Epr Spectrometer @ University of Maryland College Park
This award from the Chemistry Research Instrumentation Program will assist the Department of Chemistry at the University of Maryland in the purchase of an upgrade for an electron spin resonance (ESR) spectrometer. This upgrade is essential if the PI's are to make much more effective of the present equipment. The research projects that will be enhanced by the acquisition of this equipment include: 1) Studies of the electronic structure of organometallic 15-electron complexes, 2) Formation and electonic structure studies of 17 electron dihydride complexes, 3) Photochemical and electrochemical generation of arylnitrenium (Ar-N-R+) ions, probable intermediates in chemical carcinogenesis, and the study of their electronic structure, 4) ESR studies of organometallic and molecular-based inorganic polymers with bulk ferromagnetism and low dimensional conductivity, 5) ESR studies of enzyme-stablized radicals in biological processes. %%% An electron spin resonance (ESR) spectrometer provides information about the electronic structure of molecules and can detect the presence of "free radicals" which play an important in many chemical and biological interactions.
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0.915 |