Biological Functions of Proteinases

293 Pages·1979·7.247 MB·English

by  R. Huber (auth.), Prof. Dr. Helmut Holzer, Prof. Dr. Harald Tschesche (eds.)

30. Colloquium der Gesellschaft fUr Biologische Chemie
26.-28. April 1979 in Mosbach/Baden
Biological Functions of
Proteinases
Edited by
H. Holzer and H.Tschesche
With 142 Figures
Spri nger-Verlag
Berlin Heidelberg New York 1979
Prof. Dr. HELMUT HOLZER
Biochemisches Institut
der Albert-Ludwigs-Universitat
Hermann-Herder-StraBe 7,
7800 Freiburg/FRG
Prof. Dr. HARALD TSCHESCHE
Lehrstuhl fUr Biochemie
Fakultat fUr Chemie
der Universitat Bielefeld
UniversitatsstraBe,
4800 Bielefeld/FRG
ISBN-13:978-3-642-81397 -9 e-I SB N-13: 978-3-642-81395-5
001: 10.1007/978-3-642-81395-5
library of Congress Cataloging in Publication Data. Gesellschaft fOr Biologische Chernie. Biological functions of proteinases. Includes bibliogra
phies and index. 1. Proteinase~Congresses. 2. Protein metabolism-Congresses. I. Holzer, Helmut, 1921-. II. Tschesche, Harald. IlL Title.
QP609.P75G47. 1979.574.1'9256.79-21791.
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Softcover reprint of the hardcover 1s t edition 1979
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Preface
Proteinases were among the first enzymes to be investigated biochemi
cally, and purification and crystallization especially of proteolytic
enzymes of the digestive tract has contributed much to our present
knowledge of enzymic structure and mechanisms of catalysis. However,
for a long time little has been known about the functional aspects of
proteinases. The only exception from this have been the digestive tract
enzymes responsible for extracellular catalysis of protein breakdown
and supply of amino acids for new-protein assembly and nitrogen metab
olism in the respective organs. The work of Schoenheimer, summarized
for the first time in 1942 in a paper entitled "Dynamic state of body
constituents", showed that continuous turnover of proteins takes place
in cells. But scientists did not pay much attention to these findings
at that time. The continuous accumulation of knowledge of a variety
of intracellular proteolytic processes during the past decades has
greatly stimulated research in this field. The central role of proteo
lysis in cellular regulation has become fully evident during recent
years. It is the aim of the 30th Mosbach Colloquium to present an over
view of our present knowledge of proteinase structure, function and
control.
The relationship between globular protein structure of a proteinase
and induction of enzymic activity will be discussed for trypsin and
trypsinogen activation. One significant proteinase action is the total
degradation of proteins to serve cellular needs under different condi
tions. Thus papers to be presented will touch on general protein turn
over controlling steady-state concentrations of proteins and enhanced
degradation of proteins for cellular adaptation under starvation and
differentiation conditions in mammals and microorganisms. Other papers
will deal with degradation of "nonsense" proteins to remove harmful
protein waste, and with control of proteolysis by proteinase inhibitors
in microorganisms and mammalian tissues under normal and pathological
conditions.
Another important mode of proteinase action is the generation of nu
merous vital functions by limited proteolytic cuts. A variety of such
limited proteolytic cleavages will be discussed such as secretion and
transport of proteins across membranes, the assembly of viruses, blood
coagulation and fibrinolysis, the control of blood pressure, fertiliza
tion, the defense reaction of the complement system and the "SOS-reac
tion" upon mutagenesis in Escherichia coU. Increased proteolytic activity
is also found in transformed cells.
Even though progress in the field of proteolysis has been quite rapid
in recent years, gaps in our knowledge still exist. It is hoped that
the 30th Mosbach Colloquium, by presenting an overview of our knowledge
of the biological functions of proteinases, will allow these gaps to
be clearly recognized and then filled in by future research.
VI
.0 The urganizers are grateful to the Gesellschatt fOr Biolugische
Chemie and its chairman Prof.Dr. K. Decker for their active support of the colloquium.
Spec ial thanks are due to Prof. Dr. E. Auhagen and Prof. Dr. H. Gibian for the tech
nical organizatiod of tile meeting. The organizers are greatly indehted especially
to the Deutsche Forschungsgemeinschaft and to all persons and institutions who pro
vided the necessary funds and who helped to make this meeting a successful one.
October, 1979 HELHUT HOLZER
HARALD TSCHESCHE
Contents
Conformational Flexibility and Its Functional Significance
in Some Protein Molecules
R.HUBER (With 13 Figures)
Intracellular Protein Turnover
P.BOHLEY, H.KIRSCHKE, J.LANGNER, M.MIEHE, S.RIEMANN, Z.SALAMA,
E.SCHON, B.WIEDERANDERS, and S.ANSORGE (With 12 Figures) ....... . 17
Studies of the Pathway for Protein Degradation in
Escherichia coli and Mammalian Cells
A.L.GOLDBERG, R.VOELLMY, and K.H.SREEDHARA SWAMY
(With 5 Figures) ............................................... . 35
Lysosomes and Intracellular Proteolysis
R.T.DEAN ....................................................... . 49
Genetic and Biochemical Analysis of Intracellular
Proteolysis in Yeast
D.H.I\TOLF, C.EH~NN, and I.BECK (With 14 Figures). ............... . 55
Endogenous Inhibitors of Tissue Proteinases
J. F. LENNEY (With 5 Figures) .................................... . 73
Activity of a Rat Uterus Proteinase Inhibitor During Pregnancy
and Involution
Its Possible Importance in Control of Proteolysis in the
Myometrium
E.-G.AFTING (With 4 Figures) 87
Alkaline Proteinases in Skeletal Muscle
H.REINAUER and B.DAHLMANN (With 3 Figures) 94
Determinants in Protein Topology
G.BLOBEL (With 3 Figures) ....................................... 102
Import of Proteins into Mitochondria
N.NELSON, M.-L.MACCECCHINI, Y.RUDIN, and G.SCHATZ
(With 9 Figures) ................................................ 109
Localization and Some Properties of a Proteinase and a
Carboxypeptidase from Rat Liver
P.C.HEINRICH, R.HAAS, and D.SASSE (With 5 Figures) .............. 120
Processing of Bacteriophage Proteins
M.K.SHOWE (With 4 Figures) ...................................... 128
Proteolysis, a Determinant for Virus Pathogenicity
H.D.KLENK, F.X.BOSCH, W.GARTEN, T.KOHAMA, Y.NAGAI,
and R.ROTT (With 7 Figures) ..................................... 139
The Processing of Plasma Proteins in the Liver
G.SCHREIBER (With 9 Figures) .................................... 150
VIII
Protease Action in Carcinogenesis
W.TROLL, S. BELHAN, R. WIESNER, and C.J. SHELLA.BARGER (With 6 Figures). 165
Plasminogen Activator from Cultured Cells and from
Blood Plasma
W.-D.SCHLEUNING and A.GRANELLI-PIPERNO (With 3 Figures) ........ 171
Role of Proteinases from Leukocytes in Inflammation
M.ZIMMERMAN •.................................................... 186
Role of Granulocyte Elastase in Rheumatoid Arthritis:
Effect on Mechanical Behaviour of Cartilage.and Identification
at the Cartilage/Pannus Junction
H.MENNINGER, R.PUTZIER, W.MOHR, B.HERING, and H.D.MIERAU
(With 7 Figures) ................................................ 196
Regulation of Proteinase Activity
M. STEINBUCH ..................................................... 207
The Complement System
A.-B.LAURELL (With 5 Figures) 223
Substrate Modulation as a Control Mechanism of Plasma
Multienzyme Systems
W.VOGT (With 3 Figures) 233
Blood Coagulation
E.W.DAVIE, K.FUJIKAWA, and K.KURACHI (With 2 Figures) 238
The Kallikrein-Kinin System: A Functional Role of Plasma
Kallikrein and Kininogen in Blood Coagulation
S.IWANAGA, H.KATO, T.SUGO, N.IKARI, N.HASHIMOTO,
and S.FUJII (With 16 Figures) . '" ............................... 243
Hydrolysis of Peptide Bonds and Control of Blood Pressure
E.G.ERDC5s and T.A.STEWART (With 2 Figures) ...................... 260
Characterization of the Active Site of Angiotensin
Converting Enzyme
P.BtiNNING, B.HOLMQUIST, and J.F.RIORDAN (With 5 Figures) ........ 269
Proteolysis and Fertilization
H.FRITZ, W.MtiLLER, and A.HENSCHEN 276
Subject Index ................................................... 279
Contributors
You will find the addresses at the beginning of the respective
contribution
AFTING, E.-G. 87 LENNEY, J.F. 73
ANSORGE, S. 17 MACCECCHINI, M.-L. 109
BECK, I. 55 MENNINGER, H. 196
BELMAN, S. 1 65 MIEHE, M. 17
BLOBEL, G. 102 11IERAU, H.D. 196
BOHLEY, P. 1 7 MOHR, W. 196
BOSCH, F.X. 139 MULLER, W. 276
BUNNING, P. 269 NAGAI, Y. 1 39
DAHLMANN, B. 94 NELSON, N. 109
DAVIE, E.W. 238 PUTZIER, R. 196
DEAN, R.T. 49 REINAUER, H. 94
EHMANN, C. 55 RIEMANN, S. 1 7
ERDOS, E.G. 260 RIORDAN, J.F. 269
FRITZ, H. 276 ROTT, R. 139
FUJII, S. 243 RUDIN, Y. 109
FUJIKAWA, K. 238 SALAMA, Z. 17
GARTEN, W. 1 39 SASSE, D. 120
GOLDBERG, A.L. 35 SCHATZ, G. 109
GRANELLI-PIPERNO, A. 171 SCHLEUNING, W.-D. 171
HAAS, R. 120 SCHON, E. 17
HASHIMOTO, N. 243 SCHREIBER, G. 150
HEINRICH, P.C. 120 SHELLABARGER, C.J. 165
HENSCHEN, A. 276 SHOWE, '1. K. 1 28
HERING, B. 1 96 SREEDHARA SWAMY, K.H. 35
HOLMQUIST, B. 269 STEINBUCH, M. 207
HUBER, R. 1 STEWART, T.A. 260
IKARI, N. 243 SUGO, T. 243
IWANAGA, S. 243 TROLL, W. 165
KATO, H. 243 VOELLMY, R. 35
KIRSCHKE, H. 17 VOGT, W. 233
KLENK, H. D . 1 39 WIEDERANDERS, B. 17
KOHAMA, T. 139 WIESNER, R. 165
KURACHI, K. 238 WOLF, D.H. 55
LANGNER, J. 17 ZIMMERMAN, M. 186
LAURELL, A.-B. 223
Conformational Flexibility and Its Functional Significance
in Some Protein Molecules
R.Huber1
Introduction
The term "flexibility" in context with protein structures is used with
a variety of meanings. For instance, both a protein molecule in random
coil conformation and a molecule occurring as two different stable,
but interconvertible conformers are named flexible. A precise defini
tion of flexibility in a particular system requires determination of
the number and geometry of the various conformers, their stability,
the energy barriers separating the conformers, the kinetic parameters
of interconversion and the thermal motion of the atoms within each
conformer.
A complete analysis of the dynamic behavior of a large molecule, if
possible at all, requires a variety of physicochemical studies by dif
fraction and spectroscopic methods.
X-ray diffraction provides a static, time-averaged picture of the mol
ecule in a crystal lattice. Analysis of the crystallographic tempera
ture factor enabled by the recent development of refinement methods
(1,2), also gives some information about dynamic behavior. Spectro
scopic methods, in particular NMR, ESR, Mossbauer- and fluorescence
spectroscopy allow a much more detailed analysis in frequency in so
lution once the origin of the spectral signals is identified. This is
a difficult problem. Recent theoretical approaches (molecular dynamics
calculations) offer promising prospects of understanding the dynamic
behavior of a protein molecule (3,4).
The pancreatic trypsin inhibitor (PTI) is presently the most thorough
ly studied object by diffraction and spectroscopic techniques, as well
as molecular dynamics calculations (5,6,7,3,4). There is little doubt
that many of the phenomena observed in PTI are generally valid in pro
tein structures, but larger protein molecules show more complex behav
ior. Unfortunately spectroscopic techniques, except perhaps Mossbauer
and fluorescence spectroscopy, face serious problems with large mole
cules, so that X-ray diffraction is often the sole source of informa
tion about dynamic properties as well.
It is the intention of this article to describe the contribution of
protein crystallography to the problem of flexibility and to describe
some examples in which large-scale segmental flexibility has been dis
covered by X-ray diffraction which is apparently required for proper
function, regulation, and catalysis.
The Temperature Factor in Protein Crystallography
As the energy of lattice vibrations (phonons) is very small compared
to the energy of X-ray photons (about six orders of magnitude), lattice
IMax-Planck-Institut fur Biochemie, D-8033 Martinsried bei Munchen/FRG
2
t
E
~I!.rl-----+
_____~_f_-_-_-_-_--_-_-_-_-_l l!!..r3r _2-_-_-_-_-_-_--_-_-_-_-_-_-~_)_ ___
~( ~)~
Fig. 1. Simplified, one-dimensional energy-conformation diagram of a molecule with
a large number of rigid microstates compared to a single "soft" conformer. The space
occupied, nr3, is similar at energy E2 but becomes different at lower energy El:
nrl and nr2
vibrations cannot be observed by X-ray spectroscopy, but affect scat
tering of X-rays. The intensity of X-rays scattered by a crystal with
thermal motion compared to a perfect static crystal is reduced by an
exponential factor dependent on the scattering angle and the Debye
Waller factor, which is proportional to the mean square displacement
of an atom (8a). As X-ray scattering is an instantaneous process com
pared to lattice vibrations, it is clear that the effect of a large
number is closely related conformers (microstates), forming geometri
cally a quasi-continuum and randomly packed into a crystal lattice on
the scattered X-rays is indistinguishable from that of a single vi
brating conformer (Fig. 1). If the conformers are few and widely sep
arated in space, the formalism of mixed crystals for X-ray diffraction
holds (8a), producing a general decrease of the contributions from the
atoms affected. Cooling is the obvious means to distinguish, as only
the thermal vibrations are frozen out. This is general practice in
small molecule crystallography.
Analysis of disorder in protein crystals, however, poses several prob
lems: the experimental phases obtained by isomorphous replacement are
rather inaccurate and do not allow reliable derivation of temperature
factors. The finding that protein crystal structures can be refined
(1,2) provided a means to determine temperature factors of individual
atoms. The errors are still large compared with data obtained from
small molecule crystal structures, but the values are physically rea
sonable, when averaged for rigid groups and smoothed along the chain.
This is shown by the observation that external polypeptide loops have
higher than average temperature factors, external long amino acid side
chains show increasing temperature factors along the side chain, and
- most objectively - molecules crystallized and analyzed in different
lattices show the same trend in temperature parameters along the chain
(2,5, 9a) •