Table Of ContentSUBTILISIN ENZYMES
Practical Protein Engineering
ADV ANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
Editorial Board:
NATHAN BACK, State University of New York at Buffalo
IRUN R. COHEN, The Weizrnann Institute of Science
DAVID KRITCHEVSKY, Wistar Institute
ABEL LAJTHA, N. S. Kline Institute for Psychiatric Research
RODOLFO PAOLETTI, University of Milan
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SUBTILISIN ENZYMES
Practical Protein Engineering
Edited by
Richard Batt
Genencor International, Inc.
South San Francisco, California
and
Christian Betzel
European Molecular Biology Laboratory
Hamburg, Germany
PLENUM PRESS • NEW YORK AND LONDON
Library of Congress Cataloging-in-Publication Data
International Symposium on Subtilisin Enzymes (1992 , Hamburg.
Germany)
Subtilisin enzymes, practical protein engineering I edited by
Richard Bott and Christian Betzel.
p. cm. -- (Advances in experimental medicine and biology;
379)
"Proceedings of an International Symposium on Subtilisin Enzymes.
held September 24-26. in Hamburg. Germany"--T.p. verso.
Includes bibliographical references and index.
ISBN -13: 978-1-4613-7995-9
1. Subtilisins--Congresses. 2. Subtilisins--Inhibitors-
-Congresses. 3. Protein engineering--Congresses. I. Batt. Richard
(Richard R.) II. Betzel. Christian. III. Title. IV. Series.
OP609.S93158 1992
574. 19' 25--dc20 95-35808
CIP
Proceedings of an International Symposium on Subtilisin Enzymes,
held September 24 - 26, 1992, in Hamburg, Germany
ISBN -13: 978-1-4613-7995-9 e-ISBN-13: 978-1-4613-0319-0
DOl: 10.1007/978-1-4613-0319-0
© 1996 Plenum Press, New York
Softcover reprint of the hardcover 1st edition 1996
A Division of Plenum Publishing Corporation
233 Spring Street, New York, N. Y. 10013
1098765432 I
All rights reserved
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written
permission from the Publisher
PREFACE
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.
We would like to thank once again all those who participated in the conference and
provided manuscripts of their presentations for making the meeting and these proceedings
so substantive. We are also grateful for the generous support from Genencor International,
Novo Nordisk, Unilever, EMBL, and MAR Research.
Christian Betzel and Richard Bott
v
CONTENTS
I. Structural Studies of Subtilisin Enzymes Alone and in Complex with Inhibitors
1. The Complex between Mesentericopeptidase and Eglin-C
Z. Dauier, C. Betzel, N. Genov, and K. S. Wilson
2. Crystallographic Study of Eglin-C Binding to Thermitase . . . . . . . . . . . . . . . . . . . 5
A. Teplyakov, P. Gros, and W. G. 1. Hoi
3. Design of Specific Peptide Structures and Subtilisin Enzyme Inhibitors Using
a, J3-Dehydro-Residues .................................... '. . . . .. 11
T. P. Singh, B. Padmanabhan, P. Narula, A. K. Saxena, C. Betzel, P. Sharma,
and S. Dey
4. Structure and Function of Subtilisin BPN' as Studied through Crystallographic
Studies on a Series of Its Complexes with Genetically Engineered
Proteinaceous Inhibitor SSI ...................................... 21
T. Nonaka, T. Suzuki, N. Tanaka, S. Saito, T. Senda, K. Miura, and Y. Mitsui
5. Three Dimensional Structure of the Antibiotic Bacitracin A Complexed to Two
Different Subtilisin Proteases: Novel Mode of Enzyme Inhibition . . . . . . .. 29
S. Pfeffer-Hennig, Z. Dauter, M. Hennig, W. Hahne, K. Wilson, and
C. Betzel
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 ...... . . . . . . . .. 43
K. Hipler, 1. P. Priestle, 1. Rahuel, and M. G. Griitter
7. Crystal Strucutures of the Alkaline Proteases Savinase and Esperase from
Bacillus lentus ........................................ . . . . . . . .. 49
C. Betzel, S. Klupsch, S. Branner, and K. S. Wilson
II. New Enzymes
8. Modelling and Engineering of Enzyme/Substrate Interactions in Subtilisin-like
Enzymes of Unknown 3-Dimensional Structure ...................... 63
R.1. Siezen
vii
9. Substilases: Subtilisin-like Serine Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 75
R. J. Siezen
10. Cloning and Expression of the Gene Encoding a Novel Proteinase from
Tritirachium album Limber ....................................... 95
B. Samal, T. Boone, B. Karan, K. Chen, R. Sachdev, and T. Arakawa
III. Structure-Function Studies
11. Studies of Binding Sites in the Subtilisin from Bacillus lentus by Means of Site
Directed Mutagenesis and Kinetic Investigations ..................... 105
H. Gmn, L. M. Bech, S. B. Serensen, M. Meldal, and K. Breddam
12. Laundry Performance of Subtilisin Proteases ............................. 113
A. M. Wolff, M. S. Showell, M. G. Venegas, B. L. Barnett, and W. C. Wertz
13. Free Energy Perturbation Techniques Applied to Subtilisin BPN' Stability ..... 121
B. L. Barnett and C. B. Turner
IV. Biophysical Probes and Mutagenesis
14. Thermitase-Kinetic Differentiation to the Subtilisins ....................... 133
K. Peters, D. Bromme, G. Jahreis, and S. Fittkau
15. Fluorescence Decay of Tryptophans in Serine Proteinases from
Microorganisms: Relation to X-Ray Models ......................... 141
N. Genov, P. Nikolov, C. Betzel, and K. Wilson
16. Propeptide-Mediated Folding in Subtilisin: The Intramolecular Chaperone
Concept ...................................................... 147
U. Shinde and M. Inouye
17. Roles of the N -and C-Terminal Pro-Sequences of Aqualysin I Precursor in the
Processing and Extracellular Secretion of the Enzyme ................. 155
Y.-C. Lee, I. Terada, T. Ohta, and H. Matsuzawa
18. Crystal Structure Analysis of Subtilisin BPN' Mutants Engineered for Studying
Thermal Stability ............................................... 159
G. L. Gilliland, D. T. Gallagher, P. Alexander, and P. Bryan
19. Random Mutagenesis of the Weak Calcium Binding Site in Subtilisin Carlsberg
and Screening for Thermostability by Temperature-Gradient Gel
Electrophoresis ................................................ 171
A. Sattler, S. Kanka, W. Schrors, and D. Riesner
20. Inhibition of Proteinase K by Mercury .................................. 183
A. Muller and W. Saenger
V. Protein Engineering of Subtilisin Enzymes
21. Crystallographic Investigations of Subtilisin BPN' Mutants Engineered for
Studying Thermal Stability ....................................... 191
J. D. Oliver, T. J. Rydel, and L. C. Strickland
viii
22. Structural and Functional Consequences of Engineering the High Alkaline
Serine Protease PB92 ........................................... 203
J. M. van der Laan, O. Misset, L. J. S. M. Mulleners, G. Gerritse,
H. N. Scheifers, D. J. van Schouwen, A. V. Teplyakov, and B. W. Dijkstra
23. Engineering Surface Charges in a Subtilisin .............................. 219
M. R. Egmond, W. P. Antheunisse, P. Ravestein, A. T. A. Mooren, and
J. de Vlieg
24. pH Dependence of the Catalytic Activity ofa Subtilisin-like Proteinase ........ 229
G. Lange, C. Betzel, K. Wilson, and S. Branner
25. An Investigation of the Savinase Water Channel: Implications of Cavity
Mutations ..................................................... 235
O. H. Olsen, J. T. Pedersen, C. Betzel, S. Eschenburg, S. Branner, and
S. Hastrup
26. Substrate Specificity of Natural Variants and Genetically Engineered
Intermediates of Bacillus lentus Alkaline Proteases .................... 243
K.-H. Maurer, M. Markgraf, and D. Goddette
27. Unusual Ligand Binding at the Active Site Domain of an Engineered Mutant of
Subtilisin BL .................................................. 257
C. Paech, D. W. Goddette, T. Christianson, and C. R. Wilson
28. Studies on the Structure and Function of Subtilisin E by Protein Engineering ... 269
H. Takagi, H. Matsuzawa, T. Ohta, M. Yamasaki, and M. Inouye
29. Structural Changes Leading to Increased Enzymatic Activity in an Engineered
Variant of Bacillus lentus Subtilisin ................................ 285
R. Bott, J. Dauberman, L. Wilson, G. Ganshaw, H. Sagar, T. Graycar. and
D. Estell
Index ................................................................. 285
ix
SUBTILISIN ENZYMES
Practical Protein Engineering
