Subtilisin Enzymes, Softcover reprint of the original 1st ed. 1996 Practical Protein Engineering Advances in Experimental Medicine and Biology Series, Vol. 379

Subtilisin is the most extensively studied model system for protein engineering. The primary motivating factor for the interest in subtilisin is the commercial utility of this class of proteases. The subtilisin symposium was the first international meeting to bring together a large number of groups that have focused on the subtilisins and the subtilases-the protein superfamily of subtilisin-like enzymes. The results presented at the symposium are in this way a unique compendium of a broad spectrum of work largely focused on harnessing the potential of site-directed mutagenesis to understand and deliberately alter the function of these enzymes toward a desired end. This sort of protein engineering has been extremely successful in subtilisin, with many such "engineered" enzymes now widely used in commer­ cial enterprises. In this regard the experience derived from subtilisin does represent practical protein engineering. It is becoming clear that subtilisin represents a larger class of enzymes, the subtilases, that include many of the human pro hormone-converting enzymes. As international collabo­ rative efforts to sequence entire genomes continue, we can reasonably expect that additional members of the subtilase class will be encountered. Whenever interest in a member of this class of enzyme arises, the work on subtilisin will serve as a guide to the analysis for what in bacillus, fungi, and industry is an everyday workhorse enzyme.

I. Structural Studies of Subtilisin Enzymes Alone and in Complex with Inhibitors.- 1. The Complex between Mesentericopeptidase and Eglin-C.- 2. Crystallographic Study of Eglin-C Binding to Thermitase.- 3. Design of Specific Peptide Structures and Subtilisin Enzyme Inhibitors Using ?, ß-Dehydro-Residues.- 4. Structure and Function of Subtilisin BPN’ as Studied through Crystallographic Studies on a Series of Its Complexes with Genetically Engineered Proteinaceous Inhibitor SSI.- 5. Three Dimensional Structure of the Antibiotic Bacitracin A Complexed to Two Different Subtilisin Proteases: Novel Mode of Enzyme Inhibition.- 6. Active Site Binding Loop Stabilization in the Subtilisin Inhibitor Eglin C: Structural and Functional Studies on Specifically Designed Mutants in Complex with Subtilisin and the Uncomplexed Inhibitor.- 7. Crystal Strucutures of the Alkaline Proteases Savinase and Esperase from Bacillus lentus.- II. New Enzymes.- 8. Modelling and Engineering of Enzyme/Substrate Interactions in Subtilisin-like Enzymes of Unknown 3-Dimensional Structure.- 9. Substilases: Subtilisin-like Serine Proteases.- 10. Cloning and Expression of the Gene Encoding a Novel Proteinase from Tritirachium album Limber.- III. Structure-Function Studies.- 11. Studies of Binding Sites in the Subtilisin from Bacillus lentus by Means of Site Directed Mutagenesis and Kinetic Investigations.- 12. Laundry Performance of Subtilisin Proteases.- 13. Free Energy Perturbation Techniques Applied to Subtilisin BPN’ Stability.- IV. Biophysical Probes and Mutagenesis.- 14. Thermitase-Kinetic Differentiation to the Subtilisins.- 15. Fluorescence Decay of Tryptophans in Serine Proteinases from Microorganisms: Relation to X-Ray Models.- 16. Propeptide-Mediated Folding in Subtilisin: The IntramolecularChaperone Concept.- 17. Roles of the N- and C-Terminal Pro-Sequences of Aqualysin I Precursor in the Processing and Extracellular Secretion of the Enzyme.- 18. Crystal Structure Analysis of Subtilisin BPN’ Mutants Engineered for Studying Thermal Stability.- 19. Random Mutagenesis of the Weak Calcium Binding Site in Subtilisin Carlsberg and Screening for Thermostability by Temperature-Gradient Gel Electrophoresis.- 20. Inhibition of Proteinase K by Mercury.- V. Protein Engineering of Subtilisin Enzymes.- 21. Crystallographic Investigations of Subtilisin BPN’ Mutants Engineered for Studying Thermal Stability.- 22. Structural and Functional Consequences of Engineering the High Alkaline Serine Protease PB92.- 23. Engineering Surface Charges in a Subtilisin.- 24. pH Dependence of the Catalytic Activity of a Subtilisin-like Proteinase.- 25. An Investigation of the Savinase Water Channel: Implications of Cavity Mutations.- 26. Substrate Specificity of Natural Variants and Genetically Engineered Intermediates of Bacillus lentus Alkaline Proteases.- 27. Unusual Ligand Binding at the Active Site Domain of an Engineered Mutant of Subtilisin BL.- 28. Studies on the Structure and Function of Subtilisin E by Protein Engineering.- 29. Structural Changes Leading to Increased Enzymatic Activity in an Engineered Variant of Bacillus lentus Subtilisin.