Mechanism of proline-specific proteinases: (I) substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

Heins, Jochen; Welker, Pia; Schönlein, Chr.; Born, Ilona; Hartrodt, B.; Neubert, Klaus; Tsuru, Daisuke; Barth, A.

doi:10.1016/0167-4838(88)90067-2

Cited by 104 publications

(73 citation statements)

References 28 publications

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“…Moreover, a bulky N-terminal amino acid with free amino group (Pz position) as with Tyr or His in the peptides investigated here is optimal for high DPP-IV activity. This together with effects of the C-terminal part of the peptides might account for the relatively low K,,, and high k,, values of DPP IV for the 29-42 residue hormones GRF, GIP, amide and PHM as compared to those found earlier for small chromogenic substrates with penultimate Ala (Heins et al, 1988).…”

Section: Discussionmentioning

confidence: 89%

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Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum

Mentlein

Gallwitz

Schmidt

1993

European Journal of Biochemistry

1,094

655

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Anatomisches Institut andPeptides of the glucagodvasoactive-intestinal-peptide (VIP) peptide family share a considerable sequence similarity at their N-terminus. They either start with Tyr-Ala, His-Ala or His-Ser which might be in part potential targets for dipeptidyl-peptidase IV, a highly specialized aminopeptidase removing dipeptides only from peptides with N-terminal penultimate proline or alanine. Growthhormone-releasing factor(1-29)amide and gastric inhibitory peptidefglucose-dependent insulinotropic peptide (GIP) with terminal Tyr-Ala as well as glucagon-like peptide-1 (7 -36)amidehnsulinotropin [GLP-l(7 -36)amidel and peptide histidine methionine (PHM) with terminal His-Ala were hydrolysed to their des-Xaa-Ala derivatives by dipeptidyl-peptidase IV purified from human placenta. VIP with terminal His-Ser was not significantly degraded by the peptidase. The kinetics of the hydrolysis of GIP, GLP-1(7-36)amide and PHM were analyzed in detail. For these peptides K, values of 4-34 pM and V,,, values of 0.6-3.8 pmol . min-' . mg protein-' were determined for the purified peptidase which should allow their enzymic degradation also at physiological, nanomolar concentrations. When human serum was incubated with GIP or GLP-1(7-36)amide the same fragments as with the purified dipeptidyl-peptidase IV, namely the des-Xaa-Ala peptides and Tyr-Ala in the case of GIP or His-Ala in the case of GLP-1(7-36)amide, were identified as the main degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidylpeptidase IV, 1 mM Lys-pyrrolidide or 0.1 mM diprotin A (Ile-Pro-Ile), completely abolished the production of these fragments by serum. It is concluded that dipeptidyl-peptidase IV initiates the metabolism of GIP and GLP-1(7-36)amide in human serum. Since an intact N-terminus is obligate for the biological activity of the members of the glucagonNIP peptide family [e. g. ) is known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidylpeptidase-IV action inactivates these peptide hormones. The relevance of this finding for their inactivation and their determination by immunoassays is discussed.

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Section: Discussionmentioning

confidence: 89%

“…Small peptides or chromogenic substrates with proline in this position are far better hydrolysed than those with alanine (Heins et al, 1988). DPP IV occurs in human serum, as an ectoenzyme on the surface of capillary endothelial cells, at kidney brush-border membranes, on the…”

mentioning

confidence: 99%

Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum

Mentlein

Gallwitz

Schmidt

1993

European Journal of Biochemistry

1,094

655

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“…Because the quantitative amount of released 4-nitroaniline coincides in enzymatic and alkaline hydrolysis, the stereochemical purity of 2 is confirmed. Substrates of the dipeptide-4-nitroanilide type containing D-amino acids are known to be resistant to DP-IV-catalyzed hydrolysis [34].…”

Section: Product Analysismentioning

confidence: 99%

Influence on proline‐specific enzymes of a substrate containing the thioxoaminoacyl–prolyl peptide bond

Schutkowski

Neubert

Fischer³

1994

European Journal of Biochemistry

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Dipeptidyl peptidase IV from porcine kidney and aminopeptidase P from Escherichia coli can utilize thioxoalanyl-proline 4-nitroanilide but with decreased kinetic constants compared to the normal substrates. Product analysis showed that exclusively thioxoalanyl-proline was liberated in the case of dipeptidyl peptidase IV catalysis and thioxo-alanine in the case of aminopeptidase-P-mediated thioxo peptide bond hydrolysis. For the proline-specific aminopeptidase P the k,,/K,,, value for the thioxo peptide is 1100-fold lower than for the corresponding 0x0 peptide. This difference is entirely due to k,,,. Because the rotation about the thioxo amide bond is about 12.5 kJ mol-' more difficult than rotation about an amide bond, these data support a mechanism involving rate-limiting rotation about the scissile peptide bond. It was found that the specificity rate constant for the reaction of thioxoalanyl-proline 4-nitroanilide and dipeptidyl peptidase IV is 100 -1000-fold lower compared to the corresponding rate constant for alanyl-proline 4-nitroanilide. This remarkable effect is interpreted in terms of a distorted binding of the transition state for the thioxo substrate. The hydrolysis of the thioxo substrate by dipeptidyl peptidase IV is isomer-specific. The conformation about the nonscissile P,-PI thioxo amide bond has to be in trans for successful cleavage of the scissile peptide bond. We can now directly compare the rotational energy barrier of the prolyl peptide bond for the 0x0 and the thioxo form.Modification of the backbone of biologically active peptides has become increasingly important in the design of analogues possessing greater potency and enzymatic stability. Peptide bond modifications often induce drastic changes in flexibility of the peptide backbone. In contrast X-ray [l], infrared [2], CD [3] and NMR [4J studies of simple thioxo di-and tri-peptides have revealed that a thioxo amide mainly adopts a Z planar configuration similar to an amide, except that the thioxo-carbonyl bond is longer (C=O = 0.124 nm, C=S = 0.164 nm) [l, 51. Furthermore, the larger covalent [6] and van der Waals [7] radius of sulfur restricts the allowable $,iy angles in the vicinity of thioxo amides [8]. Thioxo peptides are also known to display a higher barrier of rotation about the C-N bond by 8-12 kJ/mol [9]. Amides and thioxo amides differ also in their hydrogen-bond forming properties. Thioxo amides are stronger acids [lo] but weaker bases than amides [ll]. Nevertheless, the introduction of a thioxo amide bond is a nearly isosteric substitution for an amide bond [12] often accompanied with surprising effects on the conformaCorrespondence to M. Schutkowski, Max-Planck-Gesellschaft zu Forderung der Wissenschaft e. V.,

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“…DPP IV is expressed ubiquitously in mammalian tissues and organs (Mentlein 1999), being located on endothelial cells of blood vessels as well as in a soluble enzyme form in blood plasma (Lodja 1979). DPP IV belongs to the prolyl oligopeptidase family of serine proteases (Barrett & Rawlings 1992) and displays a strict specificity for hydrolysing peptides from the NH 2 -terminus following a penultimate proline, alanine or hydroxyproline residue (Heins et al 1988). Therefore, in the case of GIP, DPP IV hydrolyses the NH 2 -terminal Tyr 1 -Ala 2 dipeptide, producing the truncated peptide GIP(3-42).…”

Section: Introductionmentioning

confidence: 99%

Improved biological activity of Gly2- and Ser2-substituted analogues of glucose-dependent insulinotrophic polypeptide

Gault

Flatt²,

Harriott

et al. 2003

Journal of Endocrinology

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Mechanism of proline-specific proteinases: (I) substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

Cited by 104 publications

References 28 publications

Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum

Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum

Influence on proline‐specific enzymes of a substrate containing the thioxoaminoacyl–prolyl peptide bond

Improved biological activity of Gly2- and Ser2-substituted analogues of glucose-dependent insulinotrophic polypeptide

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