ADDITIVITY BASED PREDICTION OF
BINDING TO ENZYMES

ABSTRACTS FROM OUR POSTERS

HELD AT THE

17TH SYMPOSIUM OF THE PROTEIN SOCIETY
held in Boston, MA on
July 25-30, 2003

ABSTRACT #249

In Spite of Common P1Leu Eglin c Inhibits a - Lytic Proteinase a Million Times Better Than Turkey Ovomucoid Third Domain Does M. A. Qasim1, E. L. Ash2, W. W. Bachovchin2, C. Saunders3, P. J. Ganz3 and M. Laskowski, Jr.1 1Department of Chemistry, Purdue University, West Lafayette, IN 47907, 2Department of Biochemistry, Tufts University, Boston, MA 02111, 3Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, OH 45253. a - lytic proteinase is secreted by an often renamed soil microorganism, Achromobacter enzymogens.  It is intensely studied by enzymologists and protein chemists.  Yet, in contrast to other well studied serine proteinases, little is known about its protein inhibitors except that Tufts group found that eglin c inhibits it and by the Purdue group that avian ovomucoids are largely ineffective.  As both turkey ovomucoid third domain, OMTKY3, and eglin c have P1Leu we made a great effort to measure both Ka values.  They are about 5 x 102 M-1 for OMTKY3 and 5 x 108 M-1 for eglin c.  However, P1Leu is too large for a - lytic proteinase.  Decreasing its size both for OMTKY3 and eglin c improves the binding, but the one million ratio persists.  The best binding in the P1 series is for P1Ala with Ka 6 x 104 for OMTKY3 and 7 x 1010 for eglin c.  We still do not understand this very large ratio.  For other enzymes eglin c is generally a better inhibitor but the factor is not as large.  For porcine pancreatic elastase eglin c is much worse.  (Supported at Purdue University by NIH Grant GM63539.)

 

ABSTRACT #273

Gly54 in Turkey Ovomucoid Third Domain.  A Highly Conserved, Non-Contact Residue  Lixia Wang, M. A. Qasim, S. Qasim, M. Laskowski, Jr.  Department o Chemistry, Purdue University, West Lafayette, IN  47907 Consensus variable contact residue set is obtained from structures of several serine proteinases with Kazal family inhibitors.  The remainder of the residues can be divided into 1) exposed, evolutionarily variable residues where mutations have little effect on domain stability and on enzyme binding and 2) structural, invariant or strongly conserved in evolution where the replacement will generally cause great destabilization but the effect on enzyme binding is not known.  We have just started a study of such residues by mutagenesis. Gly54 is present in 96% (371/384) of both classical and nonclassical Kazal inhibitors.  It assumes f (+91) and y (+166) angles that are allowed only for Gly or D-amino acids.  We replaced it by Glu and Ala as these residues are among those that rarely occur in natural inhibitors.  The Tm of turkey ovomucoid third domain is 73.6° and many single variants differ by only a few degrees but Tm of Glu54 OMTKY3 is 53.6° and of Ala54 OMTKY3 is 52.4°, clearly indicating a significant conformational change on replacement.  The drop in Tm made the expression of these variants especially Ala54 quite difficult.  The Ka values for interaction with 6 enzymes we study for both 54A and 54E OMTKY3 will be presented.  They differ from the wild type but in all cases by less than a factor of 10.  Black headed gull ovomucoid third domain differs from OMTKY3 at 7 positions, one of which is G54E.  Its Ka values can be predicted quite well for 5 of the 6 enzymes we study.  However, subtilisin Carlsberg is an exception.  (Supported by GM63539.)

ABSTRACT #526

Sequence to Reactivity Algorithm Helps with Understanding the Evolution of Protein Families. ZhengPing Yi1, S. Lu1, O. Vitek2, C. Bailey-Kellogg3 and M. Laskowski, Jr.1 Departments of Chemistry1, Statistics2 and Computer Sciences3, Purdue University, West Lafayette, IN 47907. In many protein inhibitors of serine proteinases the subset of positions in contact with the cognate enzyme fixes many more mutations than average for the entire molecule.  As most evolutionists think that structural and functional residues should be strongly conserved this creates a paradox.  A facile but incorrect solution is that inhibition of proteinases is not a function of the molecule but is accidental.  This makes the highly variable residues arise by pure drift – neutral mutation.  An algorithm was recently developed to predict the association standard free energies of 6 selected serine proteinases with all possible members of the Kazal proteinase inhibitor family.  Ovomucoid, which exists in all birds, consists of 3 tandem Kazal domains.  Our laboratory determined the sequences of first domains from 162, second from 23 and third from 153 species of birds.  We can therefore compare the predicted distribution function for all possible Kazal inhibitors with predicted distribution functions for the 1st, 2nd and 3rd domains of ovomucoids.  The third domains are generally very good inhibitors of the 6 selected enzymes.  Their mean standard free energy of interaction is 7 to 12 kcal/mole more negative than all possible Kazal inhibitor mean, indicating that they evolve to inhibit enzymes similar to the 6 selected enzymes.  Another problem of great interest is why the natural inhibitor sets do not contain among them inhibitors that are close in strength to the strongest possible inhibitor?  Speculative answers will be provided.  (NIH Grant GM63539.)

 

PUBLICATIONS

 

 

Our Latest Publications

 

 

Testing of the Additivity-Based Protein Sequence to Reactivity Algorithm†

 

 

 

Activity-based prediction of equilibrium constants for some protein-protein associations

 

 

 

Two Conformational States of Turkey Ovomucoid Third Domain at Low pH: Three-Dimensional Structures, Internal Dynamics, and Interconversion Kinetics and Thermodynamics

 

 

 

NMR Determination of pKa Values for Asp, Glu, His, and Lys Mutants at Each Variable Contiguous Enzyme-Inhibitor Contact Position of the Turkey Ovomucoid Third Domain

  

 

...and more

 

Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases.

 

 

 

What Can the Structures of Enzyme-inhibitor Complexes Tell Us about the Structures of Enzyme Substrate Complexes?

 

 

Interaction of Standard Mechanism, Canonical Protein Inhibitors with Serine Proteinases. M. Laskowski, Jr., M. A. Qasim, and S. M. Lu (2000) in Protein-protein recognition: The Frontiers in Molecular Biology Series (C. Kleanthous, ed.) Oxford University Press, Chapter 8.

 

 

Binding of Amino Acid Side Chains to Preformed Cavities: Interaction of Serine Proteinases with Turkey Ovomucoid Third Domains with Coded and Noncoded P1 Residues.
Arg15Lys17 -Arg18 Turkey Ovomucoid Third Domain Inhibits Human Furin.
Amino Acid Sequences of Ovomucoid Third Domains from 27 Additional Species of Birds.
A Reinvestigation of a Synthetic Peptide (TrPepz) Designed to Mimic Trypsin.
Water Molecules Participate in Proteinase-Inhibitor Interactions: Crystal Structures of Leu18, Ala18, and Gly18 Variants of Turkey Ovomucoid Inhibitor Third Domain Complexed with Streptomyces griseus Proteinase B.
Ionizable P1 Residues in Serine Proteinase Inhibitors Undergo Large pK Shifts on Complex Formation.
Probing Intermolecular Main Chain H-bonding in Serine Proteinase-Protein Inhibitor Complexes: Chemical Synthesis of Backbone Engineered Turkey Ovomucoid Third Domain.
Binding of Amino Acid Side Chains to S1 Cavities of Serine Proteinases.
Interscaffolding Additivity. Association of P1 Variants of Eglin c and of Turkey Ovomucoid Third Domain with Serine Proteinases.
Serine Protease Inhibitors, Protein in The Encyclopedia of Molecular Biology.
Expression and Characterization of Elastase Inhibitors from the Ascarid Nematodes Anisakis simplex and Ascaris suum.
Thermodynamic Criterion for the Conformation of Pa Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases.
Deleterious Effects of B -branched Residues in the S1 Specificity Pocket of Streptomyces griseus Proteinase B (SGPB): Crystal Structures of the Turkey Ovomucoid Third Domain Variants Ile18I, Val18I, and Ser18I in Complex with SGPB.

 

CURRENT COLLABORATORS
 

Prof. M.N.G. James, michael.james@ualberta.ca
Department of Biochemistry
University of Alberta
Edmonton, Alberta, Canada T6G 2H7
Ph:  (780) 492-4550

 

Prof. (Michael) Chris Laskowski, laskow@umd.edu
Department of Mathematics
417 Mathematics Building
University of Maryland
College Park, MD 20742-4015
Ph:  (301) 405-5082

 

Prof. John L. Markley, markley@nmrfam.wisc.edu
Department of Biochemistry
University of Wisconsin-Madison
420 Henry Mall
Madison, Wisconsin 53709
Ph:  (608) 263-9349
http://www.nmrfam.wisc.edu/
http://www.bmrb.wisc.edu/
http://uwstructuralgenomics.org

 

Prof. Scott A. McLuckey, mcluckey@purdue.edu
Department of Chemistry
Purdue University
1393 Brown Building
West Lafayette, IN 47907
Ph:  (765) 494-5270
http://www.chem.purdue.edu/mcluckey/index.html
http://www.chem.purdue.edu/Faculty/mcluckey.htm

 

MORE COLLABORATORS
 

Prof. Stephen Anderson, anderson@mbcl.rutgers.edu
Molecular Bio/Biochemistry
Center for Advanced Biotechnology & Medicine
Rutgers University
679 Hoes Lane West
Piscataway, NJ  08854
Ph:  (732) 235-5321

Dr. Izydor Apostol, apostol@amgen.com
Amgen, Inc.
One Amgen Center Drive, Mailstop 25-2-A
Thousand Oaks, California 91320-1799
Ph:  (805) 447-7281

 

Prof. William W. Bachovchin, wbachovc@opal.tufts.edu
Department of Biochemistry
Tufts University, Sackler
School of Graduate Biomedical Sciences
136 Harrison Avenue,
Boston, Massachusetts 02111
Ph:  (617) 636-6881

 

Prof. Wolfram Bode, bode@biochem.mpg.de
Max Planck Institut fur Biochemie
D-82152/Planegg-Martinsried
Munchen, Germany
Ph:  49 89 8578 2676
http://www.biochem.mpg.de/xray/

 

Prof. Robert Huber, huber@biochem.mpg.de
Max Planck Institut fur Biochemie
D-82152/Ann Klopferspity
Munchen, Germany
Ph:  011 4989 8578 2677 or 2688
http://www.biochem.mpg.de/xray/

 

Dr. Tomoko Komiyama, tomokomi@umich.edu
Room 5419, Med Sci 1
Dept. of Biological Chemistry
University of Michigan Med. Ctr.
Ann Arbor, MI 48109-0606
ph: 734-936-9765

 

Prof. Irwin "Tack" Kuntz, kuntz@cgl.ucsf.edu
Department of Pharmaceutical Chemistry
University of California, San Francisco
513 Parnassus / Box 0446, U80A
San Francisco, California 94143-0446
Ph:  (415) 476-1937


 

Dr. Wuyuan Lu, luw@umbi.umd.edu
Institute of Human Virology, S-516
University of Maryland, Baltimore
725 West Lombard Street
Baltimore, Maryland 21201
Ph:  (410) 706-4980

 

Prof. James McKerrow, jmck@cgl.ucsf.edu
Department of Pathology
University of California, San Francisco
Anatomical Pathology Service
DVA Medical Center 113A
San Francisco, California 94121
Ph:  (415) 476-2940
http://www.ucsf.edu/pibs/faculty/mckerrow.html

 

Prof. Michio Ogawa, mogawa@kaiju.medic.kumamoto-u.ac.jp
Department of Surgery II
Kumamoto University Medical School
1-1-1 Honjo, Kumamoto 860
Japan

 

Prof. Meredith Teilhet Morris, mmorri3@CLEMSON.EDU
Research Assistant Professor
Dept. of Genetics, Biochemistry & Life Science Studies
Biosystems Research Complex
Clemson University
Clemson, SC 29634
Ph:  (864) 656-0367

 

Prof. Jacek Otlewski, otlewski@bf.uni.wroc.pl
Institute of Biochemistry
University of Wroclaw
Tamka 2
50-137 Wroclaw, Poland
Ph:  48 71 3752 824

Prof. Jan Potempa, potempa@arches.uga.edu
Department of Biochemistry
University of Georgia
Anatomical Pathology Service
Athens, Georgia 30602
(706) 542-1713

 

Dr. Charles Saunders, saunders.cw@pg.com
The Procter & Gamble Company
Miami Valley Laboratories
P.O. Box 538708
Cincinnati, Ohio 45253
Ph:  (513) 627-2089

  

Prof. James Travis, jtravis@uga.cc.uga.edu
Department of Biochemistry
University of Georgia
Anatomical Pathology Service
Athens, Georgia 30602
Ph:  (706) 542-1713 or 1714
http://www.ega.edu/~protease/home.htm

Dr. James Wells, jawa@sunesis-pharma.com
Sunesis Pharmaceuticals, Inc.
341 Oyster Point Blvd.
South San Francisco, California 94080
Ph:  (650)-266-3500

Prof. Michael A. Wells, mawells@u.arizona.edu
Department of Biochemistry
University of Arizona
Biosciences West
Tucson, Arizona 85721
Ph:  (520) 621-3847
http://aedes.biosci.arizona.edu


 

Prof. Tadeusz Wilusz, wilusz@bf.uni.wroc.pl
Institute of Biochemistry
University of Wroclaw
Tamka 2
50-137 Wroclaw, Poland

The Data

Inhibitors of Serine Proteinases Database

P1 Variants - Data Tables

Inhibitors with Other Functions

Occurrence of Residues in the P6-P3" Region in Different Inhibitor Families

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since 05 June1998

Date Last Modified: 18 July 2002
Author: Vicki L. Booth
All Contents Copyright 1998.
Purdue University Department of Chemistry.
All Rights Reserved.