In Search of Catalytic Proficiency: The Importance of Enzyme Conformational Change to Orotidine 5'-Monophosphate Decarboxylase Catalysis PDF Download
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Author: Bryant M. Wood
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Languages : en
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The focus of this research is the role of conformational flexibility in catalysis by a TIM-barrel enzyme in pyrimidine biosynthesis, orotidine 50́9-monophosphate decarboxylase (OMPDC). OMPDC catalyzes the decarboxylation of OMP to UMP; the uncatalyzed rate for this reaction has been estimated to be 2.8 x 1016 s-1 (1). The slow rate without OMPDC is attributable to the lack of internal stabilization of the negative charge which must develop in the intermediate after decarboxylation. Because OMPDC does not utilize a cofactor in its mechanism, discovering how it is able to enhance this very slow rate to near the limits of diffusion is an important problem, kcat/KM = 1.3 x 107 M-1s-1 (2). Through alanine-scanning mutagenesis, I have identified important residues in OMPDC catalysis (3, 4). The large impact of mutating residues on the periphery of the active-site has helped develop an understanding of the importance of conformational change. Residues Ser127 and Gln185 from two different loops form an interaction that helps to coordinate loop closure with substrate binding; these residues also interact with the substrate (4). Besides active-site loop closure, crystal structures reveal the TIM-barrel of OMPDC to function as two halves which move toward one another when ligand binds. Near one of the boundaries between these two domains, I identified residues remote from the active-site which form a hydrophobic cluster in the 0́−closed0́+ state of the enzyme; Val182 from the mobile active-site loop becomes anchored in this cluster upon loop closure (3). Through site-directed mutagenesis, enzyme assays, and collaboration with X-ray crystallography experts in the Almo Group at Columbia University, I was able to determine that these hydrophobic interactions were important specifically to conformational change from an 0́−open0́+ to a 0́−closed0́+ state of the enzyme and that mutations to these residues had little impact on the 0́−closed0́+ state itself (3). It is thought that this cluster helps to coordinate the movement of domains as well as stabilize loop closure when substrate binds. Additional residues at the opposite domain interface are currently being investigated. In order to determine the rate-limiting step and to gain a better picture of the energy landscape for OMPDC catalysis, I measured the dependence of the kinetic parameters for various OMPDCs on viscosity (2). This allowed me to determine to what degree chemistry was important to the measured rate because changing viscosity affects the rate of physical steps outside of the active-site while leaving unchanged the chemical steps secluded from solvent by the active-site. For kcat and kcat/KM for yeast OMPDC and kcat/KM for the archaeal M. thermautorophicus OMPDC, OMP decarboxylation was found to be only partially dependent on the rate of chemical steps in the enzyme (2). Therefore, the rate of carbon-carbon bond cleavage, which occurs ca. 2.8 x 10-16 s-1 in solution, is enhanced by OMPDC near to the rate at which substrate can diffuse into the active site. Furthermore, kcat/KM for a 0́−faster0́+ substrate, 5-fluoroOMP (FOMP), was found to be completely dependent on viscosity for the archaeal enzyme. This demonstrated that the rate of FOMP decarboxylation is limited by the rate of FOMP diffusing into the active site. This allowed for an explanation of the small difference in the kcat/KM for OMP and FOMP, 2-fold as opposed to 1000-fold as predicted. Also, evidence for slow conformational change upon substrate binding was gleaned from the inability for FOMP decarboxylation catalyzed by the yeast enzyme to reach complete dependence on solvent viscosity. In short, because the chemical rate is far too fast and because diffusive processes will exhibit linear dependence on viscosity, there must be a viscosity sensitive conformational change in the yeast enzyme. By applying the tools of enzymology learned in the Gerlt Laboratory and working successfully with numerous collaborators, I have furthered our understanding of the mechanism of one of Nature0́9s best catalysts, OMPDC. Increasingly in enzymology, the role of conformational change in enzyme catalysis has been recognized as an important one. This research has shed light on the conformational changes that take place when substrate binds OMPDC and how the two events are coordinated.
Author: Alexander C. Roy
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Languages : en
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Author: Kurt Faber
Publisher: Springer Science & Business Media
ISBN: 3642974236
Category : Science
Languages : en
Pages : 329
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The use of natural catalysts - enzymes - for the transformation of non-natural is not at all new: they have been used for more man-made organic compounds than one hundred years, employed either as whole cells, cell organelles or isolated enzymes [1]. Certainly, the object of most of the early research was totally different from that of the present day. Thus the elucidation of biochemical pathways and enzyme mechanisms was in the foreground of the reasearch some decades ago. It was mainly during the 1980s that the enormous potential of applying natural catalysts to transform non-natural organic compounds was recognized. What started as a trend in the late 1970s could almost be called a fashion in synthetic organic chemistry in the 1990s. Although the early euphoria during the 'gold rush' in this field seems to have eased somewhat, there is still no limit to be seen for the future development of such methods. As a result of this extensive, recent research, there have been an estimated 5000 papers published on the subject [2]. To collate these data as a kind of 'super-review' would clearly be an impossible task and, furthermore, such a hypothetical book would be unpalatable for the non-expert.
Author: Charlotte W. Pratt
Publisher: Wiley
ISBN: 9781118441688
Category : Science
Languages : en
Pages : 0
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Essential Biochemistry, 3rd Edition is comprised of biology, pre-med and allied health topics and presents a broad, but not overwhelming, base of biochemical coverage that focuses on the chemistry behind the biology. Furthermore, it relates the chemical concepts that scaffold the biology of biochemistry, providing practical knowledge as well as many problem-solving opportunities to hone skills. Key Concepts and Concept Review features help students to identify and review important takeaways in each section.
Author: Benjamin A. Horwitz
Publisher: Springer Science & Business Media
ISBN: 364239339X
Category : Science
Languages : en
Pages : 394
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This volume addresses the similarities and also the differences in the genomes of soil saprophytes, symbionts, and plant pathogens by using examples of fungal species to illustrate particular principles. It analyzes how the specific interactions with the hosts and the influence of the environment may have shaped genome evolution. The relevance of fungal genetic research and biotechnological applications is shown for areas such as plant pathogenesis, biomass degradation, litter decomposition, nitrogen assimilation, antibiotic production, mycoparasitism, energy, ecology, and also for soil fungi turning to human pathogens. In addition to the model organisms Neurospora and Aspergillus, the following species are covered providing a view of pathogens and mutualists: Trichoderma, Fusarium oxysporum, Cochliobolus heterostrophus, Penicillium chrysogenum, Rhizopus oryzae, Podospora anserina, and species belonging to Agaricomycetes, Archaeorhizomycetes and Magnaporthaceae. Ecology and potential applications have guided the choice of fungal genes to be studied and it will be fascinating to follow the trends of future sequencing projects.
Author: Lizabeth A. Allison
Publisher: Wiley
ISBN: 9781118312599
Category : Science
Languages : en
Pages : 0
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Unique in in its focus on eukaryotic molecular biology, this textbook provides a distillation of the essential concepts of molecular biology, supported by current examples, experimental evidence, and boxes that address related diseases, methods, and techniques. End-of-chapter analytical questions are well designed and will enable students to apply the information they learned in the chapter. A supplementary website include self-tests for students, resources for instructors, as well as figures and animations for classroom use.
Author: Arieh Warshel
Publisher: Wiley-Interscience
ISBN: 9780471184409
Category : Science
Languages : en
Pages : 0
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This practical reference explores computer modeling of enzyme reations--techniques that help chemists, biochemists and pharmaceutical researchers understand drug and enzyme action.
Author: Peter Grunwald
Publisher: World Scientific Publishing Company
ISBN: 1783269103
Category : Science
Languages : en
Pages : 1314
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In this Completely Revised and Extended Edition with a significantly enhanced content, all Chapters have been updated considering relevant literature and recent developments until 2016 together with application oriented examples with a focus on Industrial Biocatalysis. Newly treated topics comprise among others systems metabolic engineering approaches, metagenome screening, new tools for pathway engineering, and de-novo computational design as actual research areas in biocatalysis. Information about different aspects of RNA technologies, and completely new Chapters on 'Fluorescent Proteins' and 'Biocatalysis and Nanotechnology' are also included.
Author: Lori J Goldstein
Publisher:
ISBN: 9781461526339
Category :
Languages : en
Pages : 312
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