Enzyme Structure And Mechanism Alan Fersht Pdf Download
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Protein structure and dynamics are intimately linked with biological activity. Understanding the physical basis for this activity requires knowledge of the equilibrium and kinetic properties of the protein in solution, in various free and ligand bound states. For example, conformational changes in enzymes may be crucial for optimal function and may result from subtle movements, such as the rotation of a peptide bond; from large scale fluctuations, such as the folding of regions of a protein around a prosthetic group, or from changes in the relative orientation of domains (proteins) with respect to one another in a multidomain (multiprotein) complex. Thus, understanding the interplay between structure, dynamics and function of enzymes in solution is an important goal in modern biochemistry.
Generations of students have been introduced to this subject with the first and second editions of Enzyme structure and mechanism by Alan Fersht. The detailed introductory descriptions of stopped-flow, rapid quench, steady and pre-steady state kinetics included in this text have made it a classic in graduate level courses in mechanistic enzymology. Rapid advances in protein engineering and structure elucidation over the past decade in addition to the rebirth of interest in the protein folding question require a modernized text that includes strategies and analyses of protein engineering and its effects on folding and activity. In an admirable effort, Fersht has expanded his original text to include discussions on the impact of advances in protein engineering combined with efforts directed towards understanding protein folding and catalysis. The foundation of the text is grounded on solid principles encompassing chemical reactivity, kinetics and thermodynamics in the context of the three-dimensional structure of the system under investigation.
While the scope of the book is broad, the unifying reliance on simple chemical principles in discussing enzyme mechanism and protein folding makes it a coherent work from start to finish. Thus it remains an excellent text for modern graduate courses in biochemistry and biophysics. Much of the subject matter from the original work has been retained while some chapters have been extensively updated and new sections that focus on protein stability, folding pathways and energy landscapes have been added. A particularly appealing aspect of this text is the author's writing style, which is engaging and rigorous not only in discussing the key topics of interest in protein engineering today, but also in his insistence on the importance of using correct terminology when discussing important concepts in enzymology and protein chemistry.
Protein engineering offers an approach to the modification of enzymes in terms of alteration of specificity, catalytic efficiency, pH dependence and stability. We describe how site-directed mutagenesis has been used on the tyrosyl-tRNA synthetase of Bacillus stearothermophi1 us to provide details of the energetics of individual hydrogen bonded interactions between the enzyme and its substrates. The importance of such studies is twofold. First, they give information on the mechanism of catalysis by this enzyme, and second, they indicate quantitatively how alteration of individual interactions may be used to manipulate binding and kinetic constants.
Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases. Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many fungi, in addition, excrete serine carboxypeptidases. Apparently, bacteria do not employ this group of enzymes.
It is probable that the well-known catalytic mechanism of the serine endopeptidases is also employed by the serine carboxypeptidases but presumably with the difference that the pKa of the catalytically essential histidyl residue is somewhat lower in the carboxypeptidases than in the endopeptidases. However, the leaving group specificity of these two groups of enzymes differ since the carboxypeptidases only release C-terminal amino acids from peptides (peptidase activity) and not longer peptide fragments. In addition, they release C-terminal amino acid amides (peptidyl amino acid amide hydrolase activity) or ammonia (amidase activity) from peptide amides and alcohols from peptide esters (esterase activity) and this property they share with the serine endopeptidases. Like other proteolytic enzymes the serine carboxypeptidases contain binding sites which secure the interaction between enzyme and substrate. In this laboratory, the properties of these have been studied for three serine carboxypeptidases, i.e. carboxypeptidase Y from yeast and malt carboxypeptidases I and II, by means of kinetic studies, chemical modifications of amino acid side-chains located at these binding sites and exchange of such amino acid residues by site-directed mutagenesis. 2b1af7f3a8