`BIOLOGY
`INTELLIGENCE
`UNIT
`
`. DIPEPTIDYL PEPTIDASE IV
`(CD26) IN METABOLISM
`AND THE IMMUNE REsPONSE
`
`Bernhard Fleischer
`
`'Springer
`
`R.G. LANDES CoMPANY
`
`AstraZeneca Exhibit 2149
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 41
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`
`
`MoLECUlAR
`BIOLOGY
`INTELLIGENCE
`UNIT
`
`DIPEPTIDYL PEPTIDASE IV
`( CD26) IN METABOLISM
`AND THE IMMUNE REsPONSE
`
`Bernhard Fleischer
`Bernhard Nocht Institute for Tropical Medicine
`Department of Medical Microbiology and Immunology
`Hamburg, Germany
`
`Springer
`New York Berlin Heidelberg London Paris
`Tokyo Hong Kong Barcelona Budapest
`
`R.G. LANDES COMPANY
`AUSTIN
`
`Page 2 of 41
`
`
`
`MoLEcuLAR BIOLOGY INTELLIGENCE UNIT
`
`DIPEPTIDYL PEPTIDASE IV (CD26) IN METABOLISM
`AND THE IMMUNE RESPONSE
`R.G. LANDES COMPANY
`Austin, Texas, U.S.A.
`
`Submitted: May 1995
`Published: September 1995
`
`U.S. and Canada Copyright© 1995 R.G. Landes Company
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`or
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`U.S. and Canada ISBN 1-57059-294-2
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`I
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`."•
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`, . . · .. ·. ~."\:~Iht~rnational Copyright© 199~ Springer-Verlag, Heidelberg, Germany
`''j 11
`• ·• i-
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`Allnghtsreserved.
`'·.: ·,"li
`'~
`International ISBN 3-540-60199-6
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`While the authors, editors and publisher believe that drug selection and dosage and the specifications and
`usage of equipment and devices, as set forth in this book, are in accord with current recommendations and
`practice at the time of publication, they make no warranty, expressed or implied, with respect to material
`described in this book. In view of the ongoing research, equipment development, changes in governmental
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`Library of Congress Cataloging-in-Publication Data
`
`Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response
`[edited by} Bernhard Fleischer.
`p. em. -(Molecular biology intelligence unit)
`ISBN 1-57059-294-2 (alk. paper)
`1. CD26 antigen. I. Fleischer, B. (Bernhard). II. Series.
`[DNLM: 1. Antigens, CD26-metabolism. 2. Antigens, CD26-immunology.
`QU
`136 D5965 1995}
`QR 186.6.C4D55 1995
`616.07'92-dc20
`DNLM/DLC
`for Library of Congress
`
`95-21297
`CIP
`
`~.-~.·
`~
`
`I
`I
`
`Page 3 of 41
`
`
`
`r.======= CONTENTS======;-]
`
`1. Catalytic Mechanism of Dipeptidyl Peptidase IV ....................... 1
`Hans-Ulrich Demuth, jochen Heins
`Introduction ......................................................................................... 1
`DP IV-A Serine Peptidase Exhibiting Unusual Catalytic Properties .... 2
`Mechanistic Considerations ................................................................ 17
`Conclusions ........................................................................................ 26
`
`2. Structural Organization of the DP IV Gene and Its
`Relationship with DPX and FAPa Transcripts ......................... 37
`Didier Marguet, Anne-Marie Bernard, Frederic David,
`Jciar Lazaro- Trueba, Michel Pierres
`Introduction ....................................................................................... 37
`Biochemical and Molecular Characterization of CD26 in Humans,
`Mice and Rats .................................................................................. 38
`Organization of the CD26 Gene ......................................................... 40
`Expression of DP IV ........................................................................... 43
`The DP IV-Related Proteins ............................................................... 45
`Gene Chromosomal Assignment of the DP IV-Related Family ........... 48
`Concluding Remarks .......................................................................... 49
`
`3. Functional Aspects of the Three Extracellular Domains of
`Dipeptidyl Peptidase IV: Characterization of Glycosylation
`Events, of the Collagen-Binding Site and
`of Endopeptidase Activity ......................................................... 55
`Werner Reutter, Oliver Baum, Klemens Loster, Hua Fan,
`fens Peter Bork, Kathrin Bernt, Christoph Hanski, Rudolf Tauber
`Introduction ....................................................................................... 56
`Results and Discussion ........................................................................ 58
`
`4. Control of Dipeptidyl Peptidase IV/CD26 Cell Surface
`Expression in Intestinal Cells .................................................... 79
`Germain Trugnan, Laurent Baricault, Frederic David, Martine Garcia,
`Catherine Sap in
`Introduction ....................................................................................... 79
`DP IV Cell Surface Expression as a Function ofEnterocytic
`Differentiation Is Primarily Under the Control of a
`Post-Transcriptional Event .............................................................. 82
`Metabolic and Hormonal Cell Environment May Modulate
`DP IV Cell Surface Expression ........................................................ 86
`Future Prospects ................................................................................. 92
`
`Page 4 of 41
`
`
`
`5. Modulation and Functional Diversity of Dipeptidyl
`Peptidase IV in Murine and Human Systems ........................... 99
`Brigitte Bauvois
`Introduction ....................................................................................... 99
`Regulated Expression ofDP IV ........................................................... 99
`DP IV as a Regulatory Molecule ....................................................... 102
`DP IV as a Cell Adhesion Molecule .................................................. 104
`Conclusion and Further Considerations ............................................ 106
`
`6. Role of Dipeptidyl Peptidase IV (DP IV) in Intestinal and
`Renal Absorption of Pep tides ................................................. Ill
`Matthias Brandsch, Vadivel Ganapathy, Frederick H. Leibach
`Introduction ..................................................................................... 111
`DP IV Activity in Intestinal and Renal Epithelium ........................... 112
`Peptide Substrates for DP IV in the Intestine and Kidney ................. 113
`H•/Peptide Cotransport in Intestinal and Renal Epithelium ............. 115
`Involvement of DP IV in Intestinal and Renal Peptide Absorption ... 116
`The F344 DP TV-Deficient Rat ........................................................ 118
`Peptide Hydrolysis and Peptide Transport in Intestinal and Renal
`Epithelium ofDP TV-Deficient Rats .............................................. 121
`Conclusions and Future Directions ................................................... 124
`
`7. CD26: A Key Costimulatory Molecule Involved
`in CD4 Memory T Cell Function and Activation ................... 131
`Chikao lvforimoto, Stuart F. Schlossman
`Introduction ..................................................................................... 131
`The Structure of CD26 ..................................................................... 132
`Association Molecules ofCD26 and Its Signal-Inducing Activity ...... 133
`D P IV Enzyme Activity and T Cell Activation .................................. 136
`Enhancement of Antigen-Induced T Cell Proliferation by Soluble
`CD26/DP IV ................................................................................ 136
`Possible Involvement of CD26 in HIV Infection and Apoptosis ....... 139
`Closing Remarks ............................................................................... 140
`
`8. Molecular Requirements for Signaling Through the CD26
`Molecule in T Lymphocytes ................................................... 145
`Arne von Bonin, Hans- Willi Mittriicker, Bernhard Fleischer
`Introduction ..................................................................................... 145
`Stimulation Via CD26 Is TCR Dependent ....................................... 146
`The Role ofCD3 s-Chain in the Stimulation Via CD26 .................. 147
`Role of Dipeptidyl Peptidase (DP iV) Activity in T-cell Activation ... 149
`Conclusions ...................................................................................... 150
`
`Page 5 of 41
`
`
`
`9. CD26, aT cell Accessory Molecule Induction
`of Antigen-Specific Immune-Suppression by Inactivation
`of CD26: A Clue to the AIDS Paradox? ................................. 15 5
`Meena Subrarnanyam, William B. Bachovchin, Brigitte Huber
`Role of CD26 and its Associated Enzymatic Activity
`in T cell Activation and Clonal Expansion ..................................... 156
`The CD26/HIV-1 Tat Connection .................................................. 158
`ProboroPro and HIV-1 Tat Bind to Distinct Epitopes
`of the CD26 Receptor ................................................................... 158
`Conclusions and Implications ........................................................... 159
`
`10. DP IV/CD26 on Human Lymphocytes: Functional Roles
`in Cell Growth and Cytokine Regulation ............................... 163
`Siegfried Ansorge, Frank Biihling, Tors ten Hoffmann, Thilo Kiihne,
`Klaus Neubert, Dirk Reinhold
`CD26/DP IV Is Not Only Expressed on Lymphocytes
`of the T Cell Lineage ..................................................................... 164
`Molecular Forms and Subcellular Localization ofDP IV
`Are Changed After Lymphocyte Activation .................................... 166
`DP IV-Specific Inhibitors Do Suppress DNA Synthesis
`and Cytokine Production of Lymphocytes ..................................... 168
`TGF-P1 May Play a Crucial Role in DP IV Action ........................... 170
`The Effects ofDP IV Antibodies on DNA Synthesis Depends
`on Enzymatic Activity of DP IV .................................................... 172
`DP IV and AP-N Catalyze the Hydrolysis of Cytokine Peptides
`But Not of Intact Cytokines .......................................................... 174
`DP IV Is Directly Involved in Early Events of Signal Transduction
`of Lymphocytes ............................................................................. 175
`Conclusions and Discussion .............................................................. 177
`
`11. CD26 as a Functional Marker ofTH1 Lymphocytes .............. 185
`Taila Mattern, Artur J Ulmer, Dagmar Scheel- Toellner,
`Hans-Dieter Flad
`Towards the Definition of the CD26 Antigen .................................. 185
`Expression ofCD26 (DP IV) on Human T Lymphocytes ................ 187
`CD26-A Marker of Activated T Cells, But Not Specific for
`Memory T Cells ............................................................................ 187
`Modulation of CD26 ........................................................................ 190
`Association oflnterleukin-2 Production with the Expression
`ofCD26 on Human T Lymphocytes ............................................. 192
`Differential Helper Activity ofCD4•/CD26• and CD4•/CD26- T
`Lymphocytes ................................................................................. 193
`Correlation ofCD26 Expression with TH1-Like Reactions
`in Granulomatous Diseases ............................................................ 195
`
`Index .............................................................................................. 201
`
`Page 6 of 41
`
`
`
`======CHAPTER 1 ====
`
`CATALYTIC MECHANISM
`OF DIPEPTIDYL PEPTIDASE IV
`
`Hans-Ulrich Demuth, Jochen Heins
`
`•
`
`Many regulatory peptides (i.e., neuropeptides, irrununopeptides, peptide
`
`hormones) share the common feature of having proline residues
`at positions in their sequence where they fulfill two main tasks:
`•
`Determining the properties of the secondary structures of
`the peptides necessary for their biological activity (e.g., mem(cid:173)
`brane passage, receptor binding)
`Serving as special cleavage points for proline-specific pep(cid:173)
`tidases, which are widely believed to be the processing en(cid:173)
`zymes of those peptides.
`Among these enzymes, dipeptidyl peptidase IV (DP IV) and prolyl
`endopeptidase (PEP) exhibit certain similarities in their catalytic be(cid:173)
`havior, i.e., similar second-order rate constants of substrate hydrolysis,
`preference of proline over alanine in P1-position of similar substrates
`and drastic discrimination of other amino acids at the cleavage site.
`These and other catalytic features make the proline-specific peptidases
`unique among other serine proteases.
`On the basis of structural and kinetic data as well as inhibitor
`studies we present a hypothesis on the catalytic features of post-pro(cid:173)
`line-specific peptidases.
`
`INTRODUCTION
`Peptides containing proline residues have been shown to be very
`resistant to proteolytic cleavage at these linkages. 1 Being a cyclic sec(cid:173)
`ondary amine, proline has an unique structure among natural occur(cid:173)
`ring amino acids. Many regulatory peptides contain proline residues
`determining the peptide chain conformation and biological activity. 2
`An exclusive set of proline-specific peptidases is able to regulate the
`activity of such peptides. 1•3 Among them, the dipeptidyl peptidase IV
`
`Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response, edited by
`Bernhard Fleischer.© 1995 R.G. Landes Company.
`
`Page 7 of 41
`
`
`
`2
`
`Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response
`
`(DP IV) and prolyl endopeptidase (PEP) are believed to be involved
`
`in peptide hormone processing and regulation. 1•5 An occurrence of the
`enzymes has been noted in many diseases. 6
`•7 It is believed that DP IV
`plays an important role in T-lymphocyte proliferation and might serve
`11
`as signal transmitter in transmembrane peptide transport. 8
`•
`An increasing number of authors describe protein-protein interac(cid:173)
`tions involving DP IV. DP IV (CD26) was found to be associated to
`membrane tyrosine phosphatase (CD45) on the T cell surfaceY It in(cid:173)
`teracts with matrix proteins and important regulatory proteins such as
`adenosine deamidase.J3· 15 The HIV Tat protein has been shown to be
`a binding partner of DP IV. 16
`18
`•
`Partially contradictory results, studying the impact of DP IV ac(cid:173)
`tivity on T cell growth and interleukin production using low molecu(cid:173)
`lar weight inhibitors of DP IV, have questioned the way DP IV is in(cid:173)
`volved in immune response. 18
`•19 Is the release of proline containing
`dipeptides a signal, does binding to other proteins involve signal for(cid:173)
`mation, does the protein serve as a part of a receptor/mediator com(cid:173)
`plex for peptide hormones-or all of the above?
`It is not clear yet, whether the catalytic apparatus of DP IV takes
`part in such interactions or not. In some cases mixed-type inhibition
`of ligands to DP IV was found. 18 Similarly, we have found substrate'
`inhibition and noncompetitive inhibition using artificial substrates and
`low molecular weight inhibitors.20•21 These results may be an indica(cid:173)
`tion of the existence of a secondary binding site on DP IV or the
`(catalytic) unproductive binding of ligands to the proteolytic site of
`the protein.
`However, it is unclear how the proteolytic properties and/or bind(cid:173)
`ing are related to the biological function of the protein molecule and
`nothing is known about their dependence on each other. 18•19·22 More(cid:173)
`over, even proteases exhibiting a very broad substrate specificity are
`unable to attack the peptide bonds where the prolyl residue is involved.
`(Figure 1.1 illustrates the specific cleavage points by the known pro(cid:173)
`line-specific peptidases.)
`Taking the observations from different fields of research on DP IV
`together the question arises: Is the mechanism of how proline-specific
`enzymes handle proteolysis of importance for the biological tasks of
`the proteins?23
`Thus, here we present kinetic data of the DP IV-catalyzed sub(cid:173)
`strate hydrolysis and of its inhibition, and introduce the reader to a
`hypothesis of the mechanism of DP IV and PEP on the basis of our
`research and experimental data from other laboratories, explaining the
`reasons of the unusual specificity of proline-specific peptidases by the
`proline-dependent structural flexibility of their peptide substrates.
`
`DP IV-A SERINE PEPTIDASE EXHIBITING UNUSUAL
`CATALYTIC PROPERTIES
`Checking our database we found more than 500 entries referring
`to papers dealing with dipeptidyl peptidase IV, its sources, distribu-
`
`Page 8 of 41
`
`
`
`On the Catalytic Mechanism of Dipeptidyl Peptidase IV
`
`3
`
`HIV-Protease
`
`Proline Carboxypeptidase
`
`Amino peptidase P
`
`~ 08
`t
`
`Prolidase
`
`Prolyl Carboxypeptidase
`
`00
`t Prolinase
`
`Fig. 1. 1. Proline-specific peptidases and their cleavage points.
`
`tion, purification, protein structure, biochemistry, physiology and patho(cid:173)
`physiology. This material represents a vast amount of data and infor(cid:173)
`mation about physico-chemical features, interpretations of physiologi(cid:173)
`cal properties, as well as facts and speculations about its biological
`function. In organizing these data eight main research profiles become
`obvious:
`1.
`Physiology and pathophysiology of DP IV- work dealing with
`alterations of activity and/or appearance of the protein under
`normal or pathophysiological conditions. 6•7
`2. Histochemistry and descriptive biochemistry of DP IV- ana(cid:173)
`lyzing the localization in different organisms and distribu(cid:173)
`tion in organs and tissues. 24·25
`3. Genetics- searching for DP IV genes and their expression
`from different sources, analyzing onthogenetic and phylo(cid:173)
`genetic relations.26, 27
`Biochemistry- biosynthesis, maturation, traffic, action and
`degradation of the protein. 28·29
`
`4.
`
`Page 9 of 41
`
`
`
`4
`
`Oipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response
`
`5.
`
`7.
`
`8.
`
`Immunology- research directed towards the clarification of
`16
`18
`30
`31
`the role of the enzyme in the immune system. 9
`-
`•
`•
`•
`6. Membrane biochemistry- analyzing mechanisms of peptide
`transport. 10
`.
`Protein biochemistry and recognition- studying protein-pro(cid:173)
`18
`tein interactions. 12
`-
`Enzymology- Analysis and study of the mechanism of cata(cid:173)
`lytic action of DP IV including attempts to control the
`21
`32
`33
`activity of the enzyme. 20
`•
`•
`•
`The interest of researchers from different disciplines reflects the
`impact of this particular protein on important cellular processes. However,
`profound answers on its exact manner of action are still not given.
`Our studies in the last 15 years were directed to gain insight into
`the catalytic mechanism of the enzyme and the development of spe(cid:173)
`cific inhibitors. The results of this research have to be considered to(cid:173)
`gether with the results obtained investigating prolyl endopeptidase and
`dipeptidyl peptidase II. According to our own work these enzymes behave
`catalytically very similar. Therefore we will compare data in the text
`of this chapter frequently to data obtained with PEP.
`(In the following text we will use abbreviations for kinetic param(cid:173)
`eters for comparisons as described in Table 1.1. No corrections of k-values
`will be made when data from different authors are compared. Depending
`on the conditions and the enzyme used in the different papers, the
`
`Table 1.1. Kinetic parameters
`
`Abbreviation [unit] Parameter
`
`Description
`
`KM [M]
`
`Michaelis constant
`
`kcat [sec- 1
`
`]
`
`maximum velocity
`
`kc.JKM [M- 1sec 1]
`
`specificity constant
`
`K1 [M)
`
`binding constant
`
`k;nact [sec- 1]
`
`inactivation rate
`
`k1nacJK1 [M- 1sec 1)
`
`specificity constant
`
`complex kinetic term, reflecting
`binding partially*
`calculated on the basis of the molar
`protein concentration
`also second-order rate constant,
`reflecting the bimolecular interaction
`of substrate and enzyme
`used unless otherwise noted for
`competitive inhibition, also: binding
`constant of competitive acting
`irreversible inhibitors
`first-order rate constant of
`inactivation by modifying inhibitors
`also second-order rate constant,
`reflecting the bimolecular interaction
`of inhibitor and enzyme
`
`* Due to the different localization of the rate determining steps in PEP and DP IV catalysis of
`different types of substrates, the KM-value does not reflect the simple affinity or binding
`constant. See reference 46 for details of its complex character.
`
`Page 10 of 41
`
`
`
`On the Catalytic Mechanism of Dipeptidyl Peptidase IV
`
`5
`
`activity of DP IV varies between 20 to 80 U/mg. This range of spe(cid:173)
`cific activity does not in principle influence the general tendencies
`displayed by the original data and has by definition no impact on
`inhibition constants.)
`
`SUBSTRATES AND SPECIFICI1Y OF DP IV
`
`General
`Dipeptidyl peptidase IV was discovered as a glycyl-prolyl-naphtyl(cid:173)
`amidase by Hopsu-Havu 1966.34 It is a membrane protein consisting
`of two identical subunits. DP IV was found in all kinds of living or(cid:173)
`ganisms and is distributed in mammals in virtually all organs and tis(cid:173)
`sues. Highest concentrations of the protein are localized in kidney,
`liver and in small intestine.6·7
`Depending on organism and source it carries up to 20o/o carbon
`hydrate residues, which are immunogenic but not catalytically relevant.
`Table 1.2 lists some of the enzyme's properties.
`After the primary sequences of proline-specific peptidases became
`available, 26·35·36 it was easily recognized by structural comparison, that
`the genomic organization of the enzymes is different from what was
`known about the trypsin and the subtilisin families of serine pro(cid:173)
`teases.35·37·38 Primary sequence homology between members of the family
`from one species to another species differs from 30 to 92o/o identity.39·40
`Proteins of this class are acyl peptide hydrolase, DP IV and PEP
`from animal and bacterial sources and aminopeptidase P (APP).37·41-43
`There are three crucial differences between proteins of this sub(cid:173)
`class and the other serine proteases.
`•
`They are bigger in size than the trypsin-like serine pro(cid:173)
`teases. Molecular weights range from 68 kD(APP),
`76 kD(PEP), 230 kD(DP IV) to 300 kD of the tetrameric
`acyl peptide hydrolase.4°-43
`The assumed catalytic residues Ser, His and Asp are lo(cid:173)
`cated in the C-terminal part of the primary sequence of
`the proteins.J?-44
`The arrangement of the catalytic amino acids is (i.e., in
`the DP IV gene) Ser-Asp-His, versus His-Asp-Ser of the
`trypsin-like serine pro teases. 44
`Besides phylogenetic and important structural differences, the catalytic
`mechanism as well as the specificity of the enzyme activity distinguishes
`proline-specific peptidases from other members of the serine protease
`family.
`
`•
`
`•
`
`Comparison of Specificity of DP IV and PEP
`Extensive studies in many laboratories on the substrate specificity
`of DP IV and PEP resulted in detailed information on their catalytic
`behavior. Figure 1.2 gives an overview of the substrate specificity of
`DP IV and PEP.
`
`Page 11 of 41
`
`
`
`6
`
`Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response
`
`Table 1.2. Properties of proline-specific peptidasesVAs
`
`general
`
`classification
`
`active site
`residues
`
`sequence
`around Ser
`
`occurrences
`
`activity
`optimum
`
`substrate
`specificity
`
`Dipeptidyl Peptidase IV
`(EC 3.4.14.5)
`
`Dipeptidyl Peptidase II
`{EC 3.4.14.2)
`
`Prolylendopeptidase
`(EC 3.4.21.26)
`
`glycoprotein, dimer
`M, = 230.000
`membrane bound
`
`glycoprotein, dimer
`M, = 130.000
`cytosolic
`
`monomer
`M, = 76.000
`cytosolic
`
`serine-type, subclass
`prolyl oligopeptidases
`
`serine-type, subclass
`prolyl oligopeptidases
`
`serine-type, subclass
`prolyl oligopeptidases
`
`Ser 624, Asp1o2, Hi S734
`
`serine, histidine
`
`Serss6, ASP64o, His67S
`
`Gly-T rp-Ser 624-Tyr-G ly
`
`sequence yet
`unknown
`
`Gly-Arg-Ser556-Asn-Giy
`
`rat liver,
`high concentration
`in kidney cortex,
`brush border
`pH 7.0-8.0
`
`bovine pituitary,
`rat kidney and brain,
`guinea pig testes,
`human placenta
`pH 5.0-6.0
`
`human placenta
`lamb kidney, brain,
`pig kidney, brain,
`different bacteria
`pH 7.0-8.0
`
`releases from N-termini
`of polypeptides
`H-Xaa-Pro-OH
`H-Xaa-Aia-OH
`
`releases from N-termini
`of polypeptides
`H-Xaa-Pro-OH
`H-Xaa-Aia-OH
`
`releases from oligo
`pep tides
`H-Yaa-Xaa-Pro-OH
`H-Yaa-Xaa-Aia-OH
`
`DP IV and PEP hydrolyze preferentially after proline. Both en(cid:173)
`zymes accommodate proline or alanine in P1-position. In the Prposition
`all proteinogenic amino acids may be located. All proteinogenic amino
`acids except N-methylated compounds or the amino acids proline and
`hydroxyproline may be placed in the C-terminal of the scissile peptide
`bond.6,?,zo.21.45.46 The peptide bond between amino acids in the P1- and
`Pz- position must be trans for recognition of the substratesY
`In the case of DP IV catalyzed hydrolysis, a protonated and thus
`positively charged nitrogen must be present at the N-terminus. In de(cid:173)
`tailed investigations of the pH-dependence of the DP IV- and
`PEP-catalyzed substrate hydrolysis, the participation of a general base,
`the active site histidine, could be demonstrated.20·2L46 In the case of
`the hydrolysis of substrates by DP IV, the participation of an acid
`catalyst was demonstrated. The estimated pK-values of this influence
`correspond to the pK-values of the protonated N-termini of substrates.46.4 8
`In some cases substrate inhibition of DP IV catalyzed substrate hy(cid:173)
`drolysis was observed.20·21
`In a systematic study of the DP IV proteolysis of growth hormone(cid:173)
`releasing factor and synthetic analogs, Bongers demonstrated, that in
`
`Page 12 of 41
`
`
`
`On the Catalytic Mechanism of Dipeptidyl Peptidase IV
`
`7
`
`P1-position of DP IV substrates other amino acids besides proline and
`alanine are accepted. 50 However, the difference in the specificity con(cid:173)
`stants is about three orders of magnitude (k.,./KM between 3.0 x 106
`M-1sec1-for proline in P1-position and 4.0 x 10 3 M-1sec-1-
`for leu(cid:173)
`cine in P1-position), so that physiologically the strong substrate speci(cid:173)
`ficity towards proline- and alanine- containing peptides should be rel(cid:173)
`evant. The range of reactivity estimated was Abu > Pro > Ala >> Ser >
`Val > Gly > Leu.5°
`The kinetic parameters resemble in most cases those obtained with
`artificial, chromogenic nitro anilide substrates (Tables 1.3 and 1. 5). In
`general, the nature of the side chain of the P2-amino acid has only
`weak influence on binding. Aromatic and hydrophobic aliphatic side
`chains enhance affinity by a factor of 10 but have little influence on
`k.,., (factor of 3).
`From the values compiled in Table 1.3 the substrate specificity of
`DP IV may be characterized as follows:
`•
`DP IV requires amino acids in the L-configuration in the
`P 1-position
`proline is optimally accommodated in the binding pocket
`
`•
`
`DPIV
`
`PEP
`
`0 H3N-P2-P1 !Pl '--Pn '-
`
`Gly Pro Fro-
`Ala Ala Uyp
`Val Hyp MF
`Nva Ser ~
`Leu Gly
`Ile Val
`Pro Leu
`Phe
`Ser
`Thr
`Tyr
`Lys
`Arg
`Glu
`Gin
`Asp
`Asn
`Abu
`
`-Pn--P3-P2-Pl ±Pl '--Pn '-
`!4&-
`Xaa Gly Pro
`I-lyp
`Ala Ala
`Val Hyp SaF
`Nva Sar ~a
`Leu Ala
`Ile Gly
`Pro
`Phe
`Ser
`Thr
`Tyr
`Lys
`Arg
`Glu
`Gin
`Asp
`Asn
`Abu
`
`Fig. 1.2. Substrate specificity of DP IV and PEP (minimal recognition sequence of amino acids
`numbered)
`
`Page 13 of 41
`
`
`
`8
`
`Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response
`
`•
`
`•
`
`azetidine 2-carboxylic acid as a ring size analog of proline
`fits best, exhibiting the highest catalytic rate
`• DP IV prefers aromatic and aliphatic amino acids in the L
`configuration in P 2-position
`the protonated N-terminus must be in place as in L-amino
`acids, changing its distance from the a-carbon or modifi(cid:173)
`cation by methylation results in drastic loss in binding and
`reactivity
`chemical exchange of the oxygen of the pre-proline carbo(cid:173)
`nyl group diminishes the reactivity of DP IV against the
`substrate about 3 orders of magnitude
`the physico-chemical properties of the amino acid proline
`determines the rate determining step in catalysis and not
`the electronegativity of the leaving group anilide. 51-54
`Comparing the specificity constants of substrates with increasing
`side chain length a depth of 0.5-0.6 nm for the S2-subsite was postu(cid:173)
`lated.56 In contrast to these thoughts, we found almost no significant
`influence of the specificity, studying the dependence of the hydrolytic
`constants on the length of side chain modified H-Lys (e-N-acyl)-Pro-
`4-nitroanilide derivatives (Table 1.4).
`Similar conclusions can be drawn from the data obtained using
`PEP (Table 1.5). Here the congruence of the relative reactivity of DP IV
`and PEP towards similar structured substrates should be explicitly noted.
`They hydrolyze substrates strongly stereospecifically and accommodate
`azetidine-2-carboxylic acid slightly better in the binding pocket than
`proline in P 1-position of a substrate.
`However, if the second-order rate constants are compared, DP IV
`is about a 20 times better catalyst than PEP with respect to substrates
`from both the proline and the alanine series. In contrast to DP IV no
`protonated N-terminus is essential for the catalytic activity of PEP.
`
`•
`
`•
`
`Influence of Post-translational Modifications of Substrates
`on the Catalysis by DP IV
`Recently, protein kinases have been discovered which preferentially
`recognize phosphorylation sites involving proline residues. Common
`substrate recognition motifs of the enzymes are sequences such as
`Xaa-Ser/Thr-Pro-Yaa- or -Xaa-Pro-Ser/Thr-Yaa-.59
`Such proline-directed kinases, for instance p34cdc2, p58'>'clin A and
`the mitogen-activated, proline-directed kinases p42mapk and p44"'atk, pref(cid:173)
`erentially phosphorylate serine and threonine hydroxyl groups N-terminal
`of proline residues in peptide chains. It becomes more and more evi(cid:173)
`dent, that such side chain derivatizations may be one cause of patho(cid:173)
`physiological protein modification as the abnormally hyperphosphorylated
`human Tau protein found in Alzheimer's disease.5 9-61
`Such phosphorylations/ dephosphorylations strongly influence the
`conformations of peptide chains around recognition sites and may be
`
`Page 14 of 41
`
`
`
`On the Catalytic Mechanism of Dipeptidyl Peptidase IV
`
`9
`
`Table 1.3. Comparison of the catalytic parameters of DP IV-catalyzed
`hydrolysis of 4-nitroani/ide substrates2°,21,46,5t-ss
`
`Groups of compounds
`
`k.:., [sec- 1]
`
`P2-variations, stereospecificity
`H-L-Xaa-L-Pro-4-NA
`H-D-Xaa-L-Pro-4-NA
`H-L-Xaa-D-Pro-4-NA
`H-Abu-L-Pro-4-NA
`
`1.0-10.0
`no hydrolysis
`no hydrolysis
`no hydrolysis
`
`P1-ring size variations
`H-Aia-Aze-4-NA
`H-Aia-Pro-4-NA
`H-Aia-Pip-4-NA
`
`143.0
`11.5
`3.1
`
`Position of the protonated N-terminus
`H-Aia-Pro-4-NA
`11 .5
`H-{J-Aia-Pro-4-NA
`1540.0
`H-r-Abu-L-Pro-4-NA
`3520.0
`no hydrolysis
`H-e-Ahx-L-Pro-4-NA
`
`Chemical modification of the N-terminus
`1450.0
`(CH 3) 2 -N-acetyi-Pro-4-NA
`(CH 3h -N•-acetyi-Pro-4-NA
`70,400.0
`(CH 3)2 -S+-acetyi-Pro-4-NA
`11,350.0
`
`Variation of leaving group reactivity
`H-Aia-Pro-anilides*
`H-Aia-Aia-anilides*
`
`30.0-98.0
`
`750.0 -5600.0
`
`814.0
`54.6
`2.2
`
`54.6
`6.7
`0.9
`
`0.9
`1.5
`0.3
`
`5700.0
`4770.0
`710.0
`
`4770.0
`4.4
`0.28
`
`0.613
`0.021
`0.029
`
`4500.0 - 5000.0
`0.1-20.0
`
`Replacement of carbonyl oxygen by sulfur
`11.5
`H-Aia-Pro-4-NA
`H-Aia-'P[CS-N]-Pro-4-NA
`390.0
`
`54.6
`2.8
`
`4770.0
`7.2
`
`Replacement of P,-proline
`H-L-Aia-L-Hyp-4-NA
`H-L-Xaa-L-Aia-4-NA
`H-D-Xaa-L-Aia-4-NA
`H-L-Xaa-D-Aia-4-NA
`H-Abu-L-Aia-4-NA
`
`143.0
`85.0-170.0
`9 500.0
`no hydrolysis
`8800.0
`
`72.2
`15.0-45.0
`1.0
`
`12.4
`2.6- 18.3
`0.1
`
`2.5
`
`0.28
`
`Abu - a-amino butyric acid
`r-Abu - r- amino butyric acid
`t'Ahx- £-amino hexyl acid
`Pip - pipecolic acid
`Aze- azetidine-2-carboxylic acid
`• substituents of the anilides were: -4-NO,, -4-F, -4-CI, -4-Br, -H, -4-CH,, -4-CH,CH,, -4-0CH,,
`-3-CI, -3-CH 3
`
`Page 15 of 41
`
`
`
`10
`
`Dipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response
`
`Table 1.4. Comparison of catalytic parameters of DP IV-catalyzed
`hydrolysis of various N-acyl substituted H-Lys ( c:-N-R)-Pro4-nitroanilide
`derivatives"6
`
`R
`
`KM [!lM]
`
`H
`ac