Structural Studies of Alpha-lytic Protease at Sub-Angstrom Resolution Reveal Insights Into the Mechanisms of Serine Protease Catalysis and Kinetic Stability PDF Download
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Author: Cynthia Nichole Fuhrmann Publisher: ISBN: Category : Languages : en Pages : 474
Book Description
For many decades, [alpha]-lytic protease ([alpha]LP), an extracellular bacterial protease secreted by Lysobacter enzymogenes , has served as a model in both mechanistic and structural studies for chymotrypsin-like serine proteases. In order to address questions regarding the catalytic mechanism of serine proteases, I determined three structures of [alpha]LP at ultra-high resolution: (1) the free enzyme at its active pH ([alpha]LP pH 8 ; 0.83Å resolution), (2) [alpha]LP at pH5 ([alpha]LP pH 5 ; 0.82Å resolution), and (3) [alpha]LP bound to a peptidyl boronic acid inhibitor, McOSuc-Ala-Ala-Pro-boroVal ([alpha]LP+boroVal(gol); 0.90Å resolution). The latter two structures provided analogs to the transition states of the catalytic reaction. The unexpected covalent binding of a glycerol molecule to the tetrahedral boronate in ([alpha]LP+boroVal(gol); provided the most accurate and highly-resolved model of the tetrahedral intermediate for acylation (TI 1) determined to date. Structure-specific radiation damage was apparent in electron density for these structures, and special data collection techniques were used to minimize radiation damage observed at the Ser195-boronic acid adduct. A comparison of all three structures elucidated the structural changes that occur during the first step of the catalytic reaction (ES [arrow right] TI 1 ). We propose that upon deprotonation of His57, Ser195 undergoes a conformational change that is responsible for preventing the TI 1 [arrow right] ES back-reaction. Based on precise atomic positions obtained at sub-Ångstrom resolution, we have concluded that the His57...Asp102 interaction is a standard ionic hydrogen bond in both the free enzyme and the acylation transition state, and not a low-barrier hydrogen bond as had been previously debated. Instead, I propose that transition state stabilization by chymotrypsin-like serine proteases is achieved through a network of optimized hydrogen bonds that position the catalytic triad and stabilize the Ser195-substrate tetrahedral adduct. In particular, I identified a short, ionic hydrogen bond between His57 and the amide of the substrate leaving group that may play the primary role in catalyzing the second step of the acylation reaction. A surprising outcome from these studies was the discovery of a site of conformational strain that appears to be evolutionarily linked to [alpha]LP's kinetic stability. Distortion of Phe228, a conserved residue in the protein core, is estimated to account for ~4 kcal/mol in conformational energy. The rearrangement and subsequent distortion of Phe228 by co-evolved residues surrounding it supports the hypothesis that tight packing in [alpha]LP is a key component of [alpha]LP's folding energy landscape. Subsequent mutational studies support this hypothesis, confirming our discovery of the first known functionally-relevant distortion.
Author: Cynthia Nichole Fuhrmann Publisher: ISBN: Category : Languages : en Pages : 474
Book Description
For many decades, [alpha]-lytic protease ([alpha]LP), an extracellular bacterial protease secreted by Lysobacter enzymogenes , has served as a model in both mechanistic and structural studies for chymotrypsin-like serine proteases. In order to address questions regarding the catalytic mechanism of serine proteases, I determined three structures of [alpha]LP at ultra-high resolution: (1) the free enzyme at its active pH ([alpha]LP pH 8 ; 0.83Å resolution), (2) [alpha]LP at pH5 ([alpha]LP pH 5 ; 0.82Å resolution), and (3) [alpha]LP bound to a peptidyl boronic acid inhibitor, McOSuc-Ala-Ala-Pro-boroVal ([alpha]LP+boroVal(gol); 0.90Å resolution). The latter two structures provided analogs to the transition states of the catalytic reaction. The unexpected covalent binding of a glycerol molecule to the tetrahedral boronate in ([alpha]LP+boroVal(gol); provided the most accurate and highly-resolved model of the tetrahedral intermediate for acylation (TI 1) determined to date. Structure-specific radiation damage was apparent in electron density for these structures, and special data collection techniques were used to minimize radiation damage observed at the Ser195-boronic acid adduct. A comparison of all three structures elucidated the structural changes that occur during the first step of the catalytic reaction (ES [arrow right] TI 1 ). We propose that upon deprotonation of His57, Ser195 undergoes a conformational change that is responsible for preventing the TI 1 [arrow right] ES back-reaction. Based on precise atomic positions obtained at sub-Ångstrom resolution, we have concluded that the His57...Asp102 interaction is a standard ionic hydrogen bond in both the free enzyme and the acylation transition state, and not a low-barrier hydrogen bond as had been previously debated. Instead, I propose that transition state stabilization by chymotrypsin-like serine proteases is achieved through a network of optimized hydrogen bonds that position the catalytic triad and stabilize the Ser195-substrate tetrahedral adduct. In particular, I identified a short, ionic hydrogen bond between His57 and the amide of the substrate leaving group that may play the primary role in catalyzing the second step of the acylation reaction. A surprising outcome from these studies was the discovery of a site of conformational strain that appears to be evolutionarily linked to [alpha]LP's kinetic stability. Distortion of Phe228, a conserved residue in the protein core, is estimated to account for ~4 kcal/mol in conformational energy. The rearrangement and subsequent distortion of Phe228 by co-evolved residues surrounding it supports the hypothesis that tight packing in [alpha]LP is a key component of [alpha]LP's folding energy landscape. Subsequent mutational studies support this hypothesis, confirming our discovery of the first known functionally-relevant distortion.
Author: Gary David Brayer Publisher: ISBN: Category : Streptomyces griseus Languages : en Pages : 0
Book Description
The 2.8 angstrom resolution structures of three microbial serine proteases, Strept o myces qri se us Protease A, S treptpayees qrise us Protease B and alpha lytic protease, have been determined. These enzymes are shown to be structurally related to the pancreatic family of serine proteases, rather than the previously defined bacterial subtilisin family. All three microbial enzymes are structurally similar and appear to be representative of evolutionary precursors of the mammalian pancreatic serine proteases. The determination of these structures has allowed for the detailed structural comparison of the microbial enzymes with their pancreatic counterparts. It is found that the dispositions of the active site residues Ser-214, Asp-102, His-57 and Ser-195 are nearly identical in all these enzymes. Despite the presence of only very low overall primary sequence homology (max. 21%), it is shown that approximately 60% of the residues of the microbial enzymes are topologically equivalent to residues in the pancreatic enzymes. Earlier primary sequence alignments of these enzymes were hampered by the presence of low sequence homology. A primary sequence alignment based on topological equivalence is presented. Major structural differences between the microbial and pancreatic enzymes reside in two regions. The first of these is related to the presence of a zymogen activation mechanism in the pancreatic enzymes and the apparent absence of this control mechanism in the microbial enzymes. Also observed, are significant rearrangements of polypeptide loops in the active site region of the microbial enzymes. These alterations serve to explain the unique substrate binding properties of these enzymes. Structural analyses of SGPA/peptide aldehyde complexes have also been completed. One of the peptide aldehydes investigated, chymostatin, is a naturally occurring bacterial inhibitor of serine proteases. These results show that peptide aldehydes form covalent tetrahedral hemiacetal adducts with Ser-195. The complexes formed are similar to covalent tetrahedral transition state intermediates postulated to occur during peptide catalysis. From these studies it was also possible to determine the positions of several binding subsites, which earlier investigators have shown play an important role in substrate binding and catalysis. Two SGPB/chloromethyl ketone peptide complexes were also subjects of structural analysis. These studies demonstrated that chloromethyl ketone inhibitors form two covalent bonds in the active site of SGPB. One of these is from the methylene carbon atom of the inhibitor to the imidazole ring of His-57. The other is formed from the terminal carbonyl carbon atom to the side chain of Ser-195. Comparison of inhibitor binding modes to the microbial and pancreatic serine proteases is also discussed.
Author: Bengt Nölting Publisher: Springer Science & Business Media ISBN: 354027278X Category : Science Languages : en Pages : 222
Book Description
First methods book which includes many detailed descriptions Absolutely needed and thus timely for the scientific community Comprises 15% more content and includes the mentioned special features
Author: Herbert J. Fromm Publisher: Springer Science & Business Media ISBN: 3642196233 Category : Science Languages : en Pages : 380
Book Description
This textbook, Essentials of Biochemistry is aimed at chemistry and biochemistry undergraduate students and first year biochemistry graduate students. It incorporates the lectures of the authors given to students with a strong chemistry background. An emphasis is placed on metabolism and reaction mechanisms and how they are studied. As the title of the book implies, the text lays the basis for an understanding of the fundamentals of biochemistry.
Author: Daniel John Rigden Publisher: Springer Science & Business Media ISBN: 1402090587 Category : Science Languages : en Pages : 330
Book Description
Proteins lie at the heart of almost all biological processes and have an incredibly wide range of activities. Central to the function of all proteins is their ability to adopt, stably or sometimes transiently, structures that allow for interaction with other molecules. An understanding of the structure of a protein can therefore lead us to a much improved picture of its molecular function. This realisation has been a prime motivation of recent Structural Genomics projects, involving large-scale experimental determination of protein structures, often those of proteins about which little is known of function. These initiatives have, in turn, stimulated the massive development of novel methods for prediction of protein function from structure. Since model structures may also take advantage of new function prediction algorithms, the first part of the book deals with the various ways in which protein structures may be predicted or inferred, including specific treatment of membrane and intrinsically disordered proteins. A detailed consideration of current structure-based function prediction methodologies forms the second part of this book, which concludes with two chapters, focusing specifically on case studies, designed to illustrate the real-world application of these methods. With bang up-to-date texts from world experts, and abundant links to publicly available resources, this book will be invaluable to anyone who studies proteins and the endlessly fascinating relationship between their structure and function.
Author: Stuart Hogg Publisher: John Wiley & Sons ISBN: 1119978912 Category : Science Languages : en Pages : 534
Book Description
Essential Microbiology 2nd Edition is a fully revised comprehensive introductory text aimed at students taking a first course in the subject. It provides an ideal entry into the world of microorganisms, considering all aspects of their biology (structure, metabolism, genetics), and illustrates the remarkable diversity of microbial life by devoting a chapter to each of the main taxonomic groupings. The second part of the book introduces the reader to aspects of applied microbiology, exploring the involvement of microorganisms in areas as diverse as food and drink production, genetic engineering, global recycling systems and infectious disease. Essential Microbiology explains the key points of each topic but avoids overburdening the student with unnecessary detail. Now in full colour it makes extensive use of clear line diagrams to clarify sometimes difficult concepts or mechanisms. A companion web site includes further material including MCQs, enabling the student to assess their understanding of the main concepts that have been covered. This edition has been fully revised and updated to reflect the developments that have occurred in recent years and includes a completely new section devoted to medical microbiology. Students of any life science degree course will find this a concise and valuable introduction to microbiology.
Author: Daniel Erik Otzen Publisher: John Wiley & Sons ISBN: 3527654208 Category : Science Languages : en Pages : 496
Book Description
Summing up almost a decade of biomedical research, this topical and eagerly awaited handbook is the first reference on the topic to incorporate recent breakthroughs in amyloid research. The first part covers the structural biology of amyloid fibrils and pre-fibrillar assemblies, including a description of current models for amyloid formation. The second part looks at the diagnosis and biomedical study of amyloid in humans and in animal models, while the final section discusses pharmacological approaches to manipulating amyloid and also looks at its physiological roles in lower and higher organisms. For Biochemists, Molecular Biologists, Neurobiologists, Neurophysiologists and those working in the Pharmaceutical Industry.
Author: Anne E. Osbourn Publisher: Springer Science & Business Media ISBN: 0387854983 Category : Science Languages : en Pages : 588
Book Description
Plants produce a huge array of natural products (secondary metabolites). These compounds have important ecological functions, providing protection against attack by herbivores and microbes and serving as attractants for pollinators and seed-dispersing agents. They may also contribute to competition and invasiveness by suppressing the growth of neighboring plant species (a phenomenon known as allelopathy). Humans exploit natural products as sources of drugs, flavoring agents, fragrances and for a wide range of other applications. Rapid progress has been made in recent years in understanding natural product synthesis, regulation and function and the evolution of metabolic diversity. It is timely to bring this information together with contemporary advances in chemistry, plant biology, ecology, agronomy and human health to provide a comprehensive guide to plant-derived natural products. Plant-derived natural products: synthesis, function and application provides an informative and accessible overview of the different facets of the field, ranging from an introduction to the different classes of natural products through developments in natural product chemistry and biology to ecological interactions and the significance of plant-derived natural products for humans. In the final section of the book a series of chapters on new trends covers metabolic engineering, genome-wide approaches, the metabolic consequences of genetic modification, developments in traditional medicines and nutraceuticals, natural products as leads for drug discovery and novel non-food crops.
Author: Jessup M. Shively Publisher: Springer Science & Business Media ISBN: 3540337741 Category : Science Languages : en Pages : 353
Book Description
The new series "Microbiology Monographs" begins with two volumes on intracellular components in prokaryotes. In this first volume, "Inclusions in Prokaryotes", the components, labeled inclusions, are defined as discrete bodies resulting from synthesis of a metabolic product. Research on the biosynthesis and reutilization of the accumulated materials is still in progress, and interest in the inclusions is growing. This comprehensive volume provides historical background and comprehensive reviews of eight well-known prokaryotic inclusions.
Author: Guangpu Li (Molecular biologist) Publisher: ISBN: 9781071613467 Category : Guanosine triphosphatase Languages : en Pages : 310
Book Description
This second edition volume expands on the previous edition with a discussion of new research and discoveries in the Rab field. Chapters in this book cover topics such as new information on Rab regulation and localization; interaction; function; and diseases. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Rab GTPases: Methods and Protocols, Second Edition is a valuable resource for scientists working in the fields of Rab and other small GTPases, and beyond.
Author: Cynthia Nichole Fuhrmann
Author: Cynthia Nichole Fuhrmann
Author: Gary David Brayer
Author: Bengt Nölting
Author: Herbert J. Fromm
Author: Daniel John Rigden
Author: Stuart Hogg
Author: Daniel Erik Otzen
Author: Anne E. Osbourn
Author: Jessup M. Shively
Author: Guangpu Li (Molecular biologist)