Reactions between dipeptidyl peptidase IV and diacyl hydroxylamines: mechanistic investigations

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of Kcat/Kinact. Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship. Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO2) and the decomposition of the suicide substrate in H2O and D2O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.     

Reaction of dipeptidylpeptidase IV with substrate-analogous azapeptides.

The reaction of dipeptidyl peptidase IV (EC 3.4.14.5.) with azapeptide substrates containing azaalanine or azaproline in the P1-position was investigated. Accumulation of a fairly stable acyl-enzyme could be shown for ester substrates. Ala-AzaPro-pNA is a very poor substrate of DP IV and does not accumulate an acyl-enzyme. DP IV does not react with active-site titrants for trypsin-like serine proteases.     

Reaction of Dipeptidylpeptidase IV with Substrate-Analogous Azapeptides

Abstract

The reaction of dipeptidyl peptidase IV (EC 3.4.14.5.) with azapeptide substrates containing azaalanine or azaproline in the P₁-position was investigaled. Accumulation of a fairly stable acyl-enzyme could be shown for ester substrates. Ala-AzaPro-pNA is a very poor substrate of DP IV and does not accumulate an acyl-enzyme. DP IV does not react with active-site titrants for trypsin-like serine proteases.     

Reactions Between Dipeptidyl Peptidase Iv and Diacyl Hydroxylamines: Mechanistic Investigations

Abstract

Kinetics of inactivation of dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) by N-peptidyl-O-(4-nitrobenzoyl) hydroxylamines and their enzyme-catalyzed hydrolysis were followed using independent monitoring methods, all giving similar efficiency ratios of K_cat/K_Inact

Different temperature dependences of the DP IV-inactivation and enzyme-catalyzed hydrolysis provide evidence of independent rate determining steps for both reactions. Activation parameters of inactivation are similar to those of spontaneous decomposition of the compounds, suggesting a mechanistic relationship.

Investigation of DP IV-inactivation, DP IV-catalyzed hydrolysis of N-Ala-Pro-O-Bz(4-NO₂) and the decomposition of the suicide substrate in H₂O and D₂O gave solvent isotope effects of 4.65, 2.54 and 1.02, respectively. A proton inventory of the inactivation reaction indicates involvement of more than one proton in the formation or breakdown of its transition state. The linear proton inventory found for the hydrolytic reaction is consistent with one proton transition in the rate determining step and resembles the rate limiting deacylation of Ala-Pro-DP IV. The hypothetical reaction model now locates splitting in both reactions prior to formation of a covalent intermediate during the catalytic cycle.     

Substance P in Human Plasma Is Degraded by Dipeptidyl Peptidase IV, Not by Cholinesterase

Human serum cleaves two dipeptides from the N-terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N-terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamide-gel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P-degrading activity. Vice versa, epsilon-carbobenzoxy-lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P-degrading activity. We conclude that the N-terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.     

An assay of dipeptidyl peptidase IV activity in human serum and serum of pregnant women with glycyl-L-proline-1-naphthylamide and other glycyl-L-proline-arylamides as substrates

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.     

Thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases

Aminopeptidase P (APP), dipeptidyl peptidase II (DP II), dipeptidyl peptidase IV (DP IV) and prolyl oligopeptidase (POP) are proline specific peptidases. Hence, they are able to cleave peptide bonds containing the imino acid proline. Amino acid pyrrolidides (Pyrr) and thiazolidides (Thia) are well-known product analogue inhibitors of DP IV and POP. For the first time we describe the influence of a thioxo amide bond, incorporated into these compounds, on the inhibition of the proline specific peptidases. Taking into account the substrate specificity of these peptidases, we have synthesized Xaa-psi[CS-N]-Pyrr and Xaa-psi[CS-N]-Thia of the amino acids Ala, Phe, Val and Ile. The inhibition constants were determined for the above mentioned proline specific peptidases isolated from different sources. As a result, the serine proteases DP II, DP IV and POP were inhibited competitively, whereas metal-dependent APP displayed a linear mixed-type inhibition with inhibition constants up to 10(-4) M. Thioxylation of Xaa-Pyrr and Xaa-Thia led to a slight decrease of inhibition of DP IV and POP compared to Xaa-Pyrr and Xaa-Thia, though the inhibition constants were still in the range up to 10(-7) M. As Xaa-Thia exist as two isomers, we investigated isomer specific inhibition with regard to DP IV. Thus, our studies have revealed that DP IV was only inhibited by the Z isomer of the Xaa-psi[CS-N]-Thia. For the first time, Xaa-Pyrr and Xaa-Thia were characterized as inhibitors of DP II with inhibition constants in the micromolar range. In contrast to DP IV inhibition, the Xaa-psi[CS-N]-Pyrr and Xaa-psi[CS-N]-Thia have proven to be more potent inhibitors of DP II than the corresponding Xaa-Pyrr and Xaa-Thia. Thus, these Xaa-psi[CS-N]-Thia are new potent inhibitors especially suitable for DP II with K(i) values ranging in the upper nanomolar concentration.     

Enzymes as a special class of therapeutic target: clinical drugs and modes of action

Enzymes catalyze multistep chemical reactions and achieve phenomenal rate accelerations by matching protein and substrate chemical groups in the transition state. Inhibitors that take advantage of these chemical interactions are among the most potent and effective drugs known. Recently, three new enzyme targets have been validated by FDA approval of new enzyme inhibitor drugs. These include mitogen-activated protein kinase, renin, and dipeptidyl peptidase IV. The drugs against these enzymes engage important enzyme functional groups, such as the active site serine in dipeptidyl peptidase IV. Clinical and pre-clinical discovery programs also demonstrate the same theme, as evidenced by pM and fM transition state inhibitors of purine nucleoside phosphorylase, methylthioadenosine phosphorylase, and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase, and covalent substrate trapping in leu-tRNA synthetase. The catalytic chemistry of enzymes is the key to designing potent inhibitors and makes them a special class of drug target.     

Characterization of T lymphocyte defects in patients with Hodgkin's disease using enzyme chemical markers]

Patients suffering from lymphogranulomatosis were studied with respect to cellular immune deficiencies. For this purpose, mononuclear cells from venous blood were separated and subjected to analysis of lymphocyte markers. T-lymphocytes were enumerated by means of the sheep erythrocyte (SE) rosette test. T cell subpopulations were determined using enzyme cytochemical staining for dipeptidyl peptidase IV (DP IV) and unspecific acid alphanaphthylacetate esterase (ANAE). In 18 patients with M. Hodgkin a significant reduction in the T lymphocyte count in peripheral blood was found. This T cell defect is due to a selective decrease in the TM-subpopulation as identified by enzyme cytochemical markers DP IV and ANAE (focal reaction). From these results it is concluded that patients with lymphogranulomatosis have characteristic abnormalities in the immune system in the sense of a disturbed equilibrium of immune regulatory cells.     

Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF)

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5–8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in flies but for other bioactive peptides in various invertebrates. Arch. Insect Biochem. Physiol. 37:146–157, 1998. © 1998 Wiley-Liss, Inc.     

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.     

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.     

Dipeptidyl peptidase IV inhibitors: how do they work as new antidiabetic agents?

A number of new approaches to diabetes therapy are currently undergoing clinical trials, including those involving stimulation of the pancreatic beta-cell with the gut-derived insulinotropic hormones (incretins), GIP and GLP-1. The current review focuses on an approach based on the inhibition of dipeptidyl peptidase IV (DP IV), the major enzyme responsible for degrading the incretins in vivo. The rationale for this approach was that blockade of incretin degradation would increase their physiological actions, including the stimulation of insulin secretion and inhibition of gastric emptying. It is now clear that both GIP and GLP-1 also have powerful effects on beta-cell differentation, mitogenesis and survival. By potentiating these pleiotropic actions of the incretins, DP IV inhibition can therefore preserve beta-cell mass and improve secretory function in diabetics.     

Stromal cell-derived factors 1alpha and 1beta, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8

N-terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G-protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N-terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF-1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.     

Dipeptidyl peptidase IV inhibitors: a promising new therapeutic approach for the management of type 2 diabetes

Glucagon-like peptide-1 is an insulinotropic hormone with antidiabetic potential due to its spectrum of effects, which include glucose-dependent stimulation of insulin and inhibition of glucagon secretion, tropic effects on the pancreatic beta-cells, inhibition of gastric emptying and the reduction of appetite. Glucagon-like peptide-1 is, however, extremely rapidly inactivated by the serine peptidase, dipeptidyl peptidase IV, so that the native peptide is not useful clinically. A new approach to utilise the beneficial effects of glucagon-like peptide-1 in the treatment of type 2 diabetes has been the development of orally active dipeptidyl peptidase IV inhibitors. Preclinical studies have demonstrated that this approach is effective in enhancing endogenous levels of glucagon-like peptide-1, resulting in improved glucose tolerance in glucose-intolerant and diabetic animal models. In recent studies of 3-12 months duration in patients with type 2 diabetes, dipeptidyl peptidase IV inhibitors have proved efficacious, both as monotherapy and when given in combination with metformin. Fasting and postprandial glucose concentrations were reduced, leading to reductions in glycosylated haemoglobin levels, while beta-cell function was preserved. Current information suggests dipeptidyl peptidase IV inhibitors are body weight neutral and are well tolerated. A number of dipeptidyl peptidase IV inhibitors are now in the late stages of clinical development. These have different properties, in terms of their duration of action and anticipated dosing frequency, but data from protracted dosing studies is presently not available to allow comparison of their clinical efficacy.     

Kinetic investigation of chemokine truncation by CD26/dipeptidyl peptidase IV reveals a striking selectivity within the chemokine family

Chemokines coordinate many aspects of leukocyte migration. As chemoattractants they play an important role in the innate and acquired immune response. There is good experimental evidence that N-terminal truncation by secreted or cell surface proteases is a way of modulating chemokine action. The localization of CD26/dipeptidyl peptidase IV on cell surfaces and in biological fluids, its primary specificity, and the type of naturally occurring truncated chemokines are consistent with such a function. We determined the steady-state catalytic parameters for a relevant selection of chemokines (CCL3b, CCL5, CCL11, CCL22, CXCL9, CXCL10, CXCL11, and CXCL12) previously reported to alter their chemotactic behavior due to CD26/dipeptidyl peptidase IV-catalyzed truncation. The results reveal a striking selectivity for stromal cell-derived factor-1alpha (CXCL12) and macrophage-derived chemokine (CCL22). The kinetic parameters support the hypothesis that CD26/dipeptidyl peptidase IV contributes to the degradation of certain chemokines in vivo. The data not only provide insight into the selectivity of the enzyme for specific chemokines, but they also contribute to the general understanding of CD26/dipeptidyl peptidase IV secondary substrate specificity.     

Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell-extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9-overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8-overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein-transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg-Gly-Asp integrin-binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell-extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced beta-catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling.     

Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV)

Increased concentrations of procalcitonin (PCT) are found in the plasma of patients with thermal injury and in patients with sepsis and severe infection, making this molecule important as a diagnostic and prognostic marker in these diseases. Interestingly, only the truncated form of PCT, PCT(3-116), is present in the plasma of these patients. The enzyme responsible for this truncation is unknown as yet. Here, using capillary zone electrophoresis, mass spectrometry and Edman sequence analysis, we demonstrate that dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) is capable of catalyzing the hydrolysis of PCT(1-116), releasing the N-terminal dipeptide Ala-Pro. We hypothesize that PCT(3-116) is the result of the hydrolysis of PCT(1-116) by soluble DP IV of the blood plasma or by DP IV expressed on the surface of cells.     

Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes.

Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma-glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid. It is suggested that the intestinal brush border peptidases, dipeptidyl peptidase IV and gamma-glutamyltransferase, are closely related to the corresponding enzymes obtained from the kidney.