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Are diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?
Biochimica e: BiophysicaActa, 1076(1991)314-316 © 1991 ElsevierSciencePublishersB.V,(BiomedicalDivision)0167-4838/91/$03.50 ADONIS 016748389100098R
314
BBAPRO30278
BBA Report
Are diprotin A (lie-Pro-lie) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV? J. R a h f e l d 1, M. S c h i e r h o r n 2, B. H a r t r o d t l, K. N e u b e r t i a n d J. H e i n s l I Martin-Luther-University, BiozechnikumHalle and "M~ir.. Luther-Universit): Deparzmemof Chemistry Halle (F.R. G.)
(Received17 July 1990)
Key words: Dipcptidylpeptidas¢IV: Substratespccifidtyinhibition Dipeptidyi peptidase IV preferably hydrolyzes peptides and proteins with a penultimate proline residue. Umezawa and co-workers (Umezawa et al. (1984) J. Antibiotics 37, 422-425) reported that diprotin A (lie-Pro-lie) and diprotin B (Val.Pro-Leu) are inhibitors for dipepfidyl peptidase IV. We could show that both compounds as well as other tripepfides with a penultimate proline residue are substrates for dipepfidyl peptidase IV. An apparent competitive inhibition by those compoonds is a Kinetic artifact due to the substrate-like structure of such tripeptides.
Dipeptidyi peptidase IV (EC 3.4.14.5) is a membrane-bound proline.specific exopeptidase of various mammalian tissues [1]. We investigated in detail the substrate specificity of this enzyme [2], which may be summarized in the following way: (1) Dipeptidyl peptidase IV hydrolyzes oligopeptides and proteins from the N-terminus, splitting off dipeptide units, when the penultimate residue is proline, hydroxyproline, dehydroproline, pipecolic acid or alanine. The best substrates according :o their kcat/K m values are those with a proline residue in the Pl-position. (2) DP IV requires a "trans" peptide-bond between P1 and P2 residues [31. (3) The N-terminal amino group of substrates must be protonated in order to be susceptible to DP IV [4]. (4) A proline residue in the Pl'-position of substrates prevents substrate hydrolysis by dipeptidyl peptidase IV. This enzyme does not release arginylproline from bradykinin [5]. Umezawa and co-workers [61 found that diprotin A (lie-Pro-lie) and diprotin B (Val-Pro-Leu) are inhibitors of dipeptidyl peptidase IV. This result is rather surprising since both compounds have the overall substratelike structure for DP IV. Th,- aim of the present publication was to clarify the question of whether diprotin A and B are substrates or inhibitors for dipeptidyl peptidase IV.
CorrespondL~nce:J. Heins, Martin-Luther-University. Biotechnikum, Weinbergweg~.,5a,Halle/S. 4050, F.R.G.
The analysis of the DP iV-catalyzed hydrolysis of lie-Pro-lie by HPLC (Table I) shows that this tripeptide is completely hydrolyzed by dipeptidyl peptidase IV yielding isoleucylproline and isoleacine. Diprotin B (Val-Pro-Leu) and other similar peptides with a penultimate proline residue are also substrates for DP IV (data not shown). For the accurate estimation of the catalytic constants of DP IV-catalyzed hydrolysis of tripeptides, such as diprotin A and diprotin B, we developed a spectrophotometric method, where the hydrolysis-reaction could be followed continuously. The hydrolysis of Val-Pro-Leu and other tripeptides by DP IV yields a time-dependent decrease in the
TABLE 1 Hydrolysis of lie-Pro-lie by dipeptid¥1peplidase IV, followed by HPLC (peak area in ~)
(Retention time: lie-Pro-OH,2.5 rain; lie-Pro-lie,20 rain) Experimental conditions: (1) Incubation mixture. Enzyme concentration. 4.7, 10-7 M; lie-Pro-lie. 2.66 raM; and buffet, see Fig. 1. (2) HPLC: elucnt, (A) acetonittile; (B) KH2PO4 10 mM, pH 3 (H3PO4), A:B (10:90). Column: LiChroCART4-4, LiChrospher 100 RP-18 5 pm LiChroCart 125-4,LiChrospher100 RP-185 pm, UV-detector([AO~, MERCK) 220 nm. Time
lie-Pro-OH (%)
lie-Pro-Re (%)
5 rain 36 rain 70rain 20.5 h
2 37 66 100
98 63 34 0
315
"~5 E - 02~
[
~ o~
~ Z PC
50Z 0 3 ~
i
01 ~ , ~ ~ ~ , , , t A i , I , i i I I l 0 20E ' ' 40E 80E 80E ~OE Substsole {M) Fig. I. Dependenceof DP IV-cat,alM hydrolysis of Val-Pro-Le- on substrate concentration. Experimental conditions: enzyme concentration, 4.7-10 -9 M, 39 mM sodium phosphat,' buffer (pH 7.6); ionic strength (KCi). 0.125, t = 30°C). The data represent mean and standard deviation (n = 3-5) and the curve is calculated by a nontinear regression program (values for k~,, K= and k~,/Km, see Table IlL
absorbance between 200 and 212 nm and an increase of the absorbance between approx. 216 and 250 nm. The DP IV-catalyzed hydrolysis of lie-Pro-lie and Val-Pro-Leu was followed at 206 nm. The extinction coefficient at this wavelength was estimated as 3000 __ 150 cm2- mol -t. The velocity of the hydrolysis of ValPro-Lcu by dipeptidyl pcptidase IV shows typical Michaelis-Menten kinetics (Fig. 1). The catalytic constants for the hydrolysis of several tripeptides, including diprotin A and B, in comparison to the corresponding dipeptide-4-nitroanilides are shown in Table ii.
TABLE II
DP IV-catalyzed hydrolysis of aripeptides and dipeptide.4mitroanilides Exp¢rimental conditions: enzyme concentration, 4.7.10 -9 M: substrate concentration, between 0.5.K m and lO.Km; 39 raM sodium phosphate-buffer (pH 7.6); ionic strength (KCI) 0.125, t = 30°C. The data represent results from a nonfincar ~ i o n program (Fit to Michadis-Mcnten equation, regression patmncter and S.E. for kea t and K= and derived for kcat/K m considering error propagation). For each substrate concentration at least three measurements were made. Compound
k~., (s-tl
K.. t0 $ (M)
kca, / K m"10- 5
Pro-Pro-Lcu
Pro-Pro-Ala Pro-Pro-pNA Val-Pro-Leu
24.9:t:5.3 22.0+1.2 51.54-0.3 27.04-1.6
1.0 1.7 0.9 1.6
25 13 56 17
Val-Pro-pNA Tyr-Pro-lle Tyr-Pro-Phe Tyr-Pro-pNA lie-Pro-lie Ile-Pro-pNA
44.9±0.9 2.6±0.1 27.04-i.8 62.9±2.2 1.3±0.1 28.54-4.9
1.3 ±0.1 35 ±2 0.45±0.01 5.7+0.1 6.6 4-0.7 4.24-0.5 4.0 ±0.3 16 4-1 0.40±0.05 3.44-0.4 1.2 4-0.7 23 + 4
4-0.1 +0.1 4-0.1 4-0.1
(s-t.M -|) +6 +1 +2 +!
- 20E
O
20E
40E
Io (M)
Fig. 2. Apparent inhibition of the DP IV-catalyzed hydrolysis of Ala-Pro-pNA by the tripeptide Val-Pro-Lcu. Experimental conditions: enzyme concentration, 4.7-10 -~ M: Ala-Pro-pNA, 1.56-10 - s M, 3.11-10 -s M, 6.23-10 - s M, 1.56-10 -4 M: 39 mM sodium phosphate buffer (pH 7.6): ionic strength (KC~}, 0.125, t = 3 0 ° C . The Dixon plot was analyzed using a linear regression program. The apparent K i value of Val-Pro-Leu toward DP IV-catalyzed hydrolysis of Ala-Pro-pNA is (1.9±0.4)-10 -4 M (calculated as mean and standard deviation from all possibte intersections of the straight lines).
The data of Table II show that the value of k ~ , / K m of DP IV-catalyzed hydrolysis of tripeptides is generally lower than that for analogous dipeptide-4-nitroanifides. This is also true for the corresponding value of kcar In the case of tripeptides with an isoleucine residue in the Pl'-position, we found a remarkably low value of k~at and K m in comparison to other substrates. It is known that the rate.limiting step for the hydrolysis of substrates with a proline residue in the Pl-position is the deacylation reaction [7], which means that the k~t value is independent of the residue in the Pl'-position. This holds true, at least for substrates of the structure X-Pro-Y (X: amino acid; Y: arylamide). For natural substrates, where Y is any amino acid or peptide, we do not yet know the nature of the rate-limiting step of the hydrolysis of those compounds by dipeptidyi pcptidase IV. Considering the differences in the kea, values of the hydrolysis of tripeptid~ and dipeptide-4-nitroanilides, it may be suggested that with tripeptides the rate-limiting step of DP IV-catalyzed hydrolysis is at least partially the acylation reaction. Our results of the hydrolysis of tripeptides by dipeptidyl peptidase iV clearly show that those compounds without any doubt are substrates of DP IV. When the DP IV-catalyzed hydrolysis of a good substrate (e.g., Ala-Pro-pNA) is followed in the presence of a tripeptide (e.g., Val-Pro-Leu), the tripeptide leads to the picture of a kinetic competitive inhibition (Fig. 2). It can be shown by deriving the rate equation for the situation where one enzyme hydrolyzes two substrates
316 simultaneously that one substrate ($2) formally acts as the competitive inhibitor for the other substrate ($1) (e.g., SI: Aia-Pro-pNA; $2: Val-Pro-Leu). The Ki value in this case is not the true thermodynamic dissociation constant of the E-S2-complex but the K m value for the substrate $2, which is in the general case a complex kinetic constant. The g m value of the hydrolysis of Val-Pro-Leu by D P IV was estimated as (1.6 _+ 0.1)10 -5 M and the apparent K i value of the same compound towards the DP IV-catalyzed hydrolysis of AlaPro-plqA was (1.9 ± 0.4)-10 -5 M and in the case of Ala-AiaopNA as substrate (1.7 +_ 0.2). 10 -5 M, respectively. These values are identical and a support for the above mentioned suggestion. In conclusion it is appropriate to refer to diprotin A, diprotin B, as well as to other tripeptides with an analogous structure as substrates for dipeptidyl peptidase IV, An apparent competitive inhibition of DP IV
by those compounds is an interesting kinetic artifact which comes from the substrate-like nature of tripeptides with a penultimate proline residue.
References 1 Waher, R., Simmons. W.H. and Ynshimoto, T. (1980) Mol. Cell. Bio. 30. 111-127. 2 Heins. J. Welker, P., Sch~nlein. Chl. Born. L. Harlrodt. B.. Neubert, K~ Tsuru. D. and Barlh, A. (1988) Biochim. Biophys. Acta 954, 161-169. 3 Fischer, G. Heins, J. and Barth, A. (1983) Biochim. Biophys. Acta 742, 452-462. 4 Hein~ J, Neubet't, K., Scha~'weiB, B, and Barth, A. (1983) Federation Proceedings 42, 1324. 5 Kato, T~ Nagalsu, T., Fukasawa, K., Harada, M., Nagatsu, 1. and Sakakibant, S. (1978} Biochim. Biophys. Acta 525, 417-422, 6 Umezawa, H, Aoyagi. T., Ogawa, K., Naganawa, li., Hamada. M. and Takeuchi, T. (1984} J. Antibiotics 37, 422-425. 7 Wolf, B., F'~cl~r, G. and Barth, A. 0978) Acla Biol. Med. Germ. 37. 409-420.
314
BBAPRO30278
BBA Report
Are diprotin A (lie-Pro-lie) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV? J. R a h f e l d 1, M. S c h i e r h o r n 2, B. H a r t r o d t l, K. N e u b e r t i a n d J. H e i n s l I Martin-Luther-University, BiozechnikumHalle and "M~ir.. Luther-Universit): Deparzmemof Chemistry Halle (F.R. G.)
(Received17 July 1990)
Key words: Dipcptidylpeptidas¢IV: Substratespccifidtyinhibition Dipeptidyi peptidase IV preferably hydrolyzes peptides and proteins with a penultimate proline residue. Umezawa and co-workers (Umezawa et al. (1984) J. Antibiotics 37, 422-425) reported that diprotin A (lie-Pro-lie) and diprotin B (Val.Pro-Leu) are inhibitors for dipepfidyl peptidase IV. We could show that both compounds as well as other tripepfides with a penultimate proline residue are substrates for dipepfidyl peptidase IV. An apparent competitive inhibition by those compoonds is a Kinetic artifact due to the substrate-like structure of such tripeptides.
Dipeptidyi peptidase IV (EC 3.4.14.5) is a membrane-bound proline.specific exopeptidase of various mammalian tissues [1]. We investigated in detail the substrate specificity of this enzyme [2], which may be summarized in the following way: (1) Dipeptidyl peptidase IV hydrolyzes oligopeptides and proteins from the N-terminus, splitting off dipeptide units, when the penultimate residue is proline, hydroxyproline, dehydroproline, pipecolic acid or alanine. The best substrates according :o their kcat/K m values are those with a proline residue in the Pl-position. (2) DP IV requires a "trans" peptide-bond between P1 and P2 residues [31. (3) The N-terminal amino group of substrates must be protonated in order to be susceptible to DP IV [4]. (4) A proline residue in the Pl'-position of substrates prevents substrate hydrolysis by dipeptidyl peptidase IV. This enzyme does not release arginylproline from bradykinin [5]. Umezawa and co-workers [61 found that diprotin A (lie-Pro-lie) and diprotin B (Val-Pro-Leu) are inhibitors of dipeptidyl peptidase IV. This result is rather surprising since both compounds have the overall substratelike structure for DP IV. Th,- aim of the present publication was to clarify the question of whether diprotin A and B are substrates or inhibitors for dipeptidyl peptidase IV.
CorrespondL~nce:J. Heins, Martin-Luther-University. Biotechnikum, Weinbergweg~.,5a,Halle/S. 4050, F.R.G.
The analysis of the DP iV-catalyzed hydrolysis of lie-Pro-lie by HPLC (Table I) shows that this tripeptide is completely hydrolyzed by dipeptidyl peptidase IV yielding isoleucylproline and isoleacine. Diprotin B (Val-Pro-Leu) and other similar peptides with a penultimate proline residue are also substrates for DP IV (data not shown). For the accurate estimation of the catalytic constants of DP IV-catalyzed hydrolysis of tripeptides, such as diprotin A and diprotin B, we developed a spectrophotometric method, where the hydrolysis-reaction could be followed continuously. The hydrolysis of Val-Pro-Leu and other tripeptides by DP IV yields a time-dependent decrease in the
TABLE 1 Hydrolysis of lie-Pro-lie by dipeptid¥1peplidase IV, followed by HPLC (peak area in ~)
(Retention time: lie-Pro-OH,2.5 rain; lie-Pro-lie,20 rain) Experimental conditions: (1) Incubation mixture. Enzyme concentration. 4.7, 10-7 M; lie-Pro-lie. 2.66 raM; and buffet, see Fig. 1. (2) HPLC: elucnt, (A) acetonittile; (B) KH2PO4 10 mM, pH 3 (H3PO4), A:B (10:90). Column: LiChroCART4-4, LiChrospher 100 RP-18 5 pm LiChroCart 125-4,LiChrospher100 RP-185 pm, UV-detector([AO~, MERCK) 220 nm. Time
lie-Pro-OH (%)
lie-Pro-Re (%)
5 rain 36 rain 70rain 20.5 h
2 37 66 100
98 63 34 0
315
"~5 E - 02~
[
~ o~
~ Z PC
50Z 0 3 ~
i
01 ~ , ~ ~ ~ , , , t A i , I , i i I I l 0 20E ' ' 40E 80E 80E ~OE Substsole {M) Fig. I. Dependenceof DP IV-cat,alM hydrolysis of Val-Pro-Le- on substrate concentration. Experimental conditions: enzyme concentration, 4.7-10 -9 M, 39 mM sodium phosphat,' buffer (pH 7.6); ionic strength (KCi). 0.125, t = 30°C). The data represent mean and standard deviation (n = 3-5) and the curve is calculated by a nontinear regression program (values for k~,, K= and k~,/Km, see Table IlL
absorbance between 200 and 212 nm and an increase of the absorbance between approx. 216 and 250 nm. The DP IV-catalyzed hydrolysis of lie-Pro-lie and Val-Pro-Leu was followed at 206 nm. The extinction coefficient at this wavelength was estimated as 3000 __ 150 cm2- mol -t. The velocity of the hydrolysis of ValPro-Lcu by dipeptidyl pcptidase IV shows typical Michaelis-Menten kinetics (Fig. 1). The catalytic constants for the hydrolysis of several tripeptides, including diprotin A and B, in comparison to the corresponding dipeptide-4-nitroanilides are shown in Table ii.
TABLE II
DP IV-catalyzed hydrolysis of aripeptides and dipeptide.4mitroanilides Exp¢rimental conditions: enzyme concentration, 4.7.10 -9 M: substrate concentration, between 0.5.K m and lO.Km; 39 raM sodium phosphate-buffer (pH 7.6); ionic strength (KCI) 0.125, t = 30°C. The data represent results from a nonfincar ~ i o n program (Fit to Michadis-Mcnten equation, regression patmncter and S.E. for kea t and K= and derived for kcat/K m considering error propagation). For each substrate concentration at least three measurements were made. Compound
k~., (s-tl
K.. t0 $ (M)
kca, / K m"10- 5
Pro-Pro-Lcu
Pro-Pro-Ala Pro-Pro-pNA Val-Pro-Leu
24.9:t:5.3 22.0+1.2 51.54-0.3 27.04-1.6
1.0 1.7 0.9 1.6
25 13 56 17
Val-Pro-pNA Tyr-Pro-lle Tyr-Pro-Phe Tyr-Pro-pNA lie-Pro-lie Ile-Pro-pNA
44.9±0.9 2.6±0.1 27.04-i.8 62.9±2.2 1.3±0.1 28.54-4.9
1.3 ±0.1 35 ±2 0.45±0.01 5.7+0.1 6.6 4-0.7 4.24-0.5 4.0 ±0.3 16 4-1 0.40±0.05 3.44-0.4 1.2 4-0.7 23 + 4
4-0.1 +0.1 4-0.1 4-0.1
(s-t.M -|) +6 +1 +2 +!
- 20E
O
20E
40E
Io (M)
Fig. 2. Apparent inhibition of the DP IV-catalyzed hydrolysis of Ala-Pro-pNA by the tripeptide Val-Pro-Lcu. Experimental conditions: enzyme concentration, 4.7-10 -~ M: Ala-Pro-pNA, 1.56-10 - s M, 3.11-10 -s M, 6.23-10 - s M, 1.56-10 -4 M: 39 mM sodium phosphate buffer (pH 7.6): ionic strength (KC~}, 0.125, t = 3 0 ° C . The Dixon plot was analyzed using a linear regression program. The apparent K i value of Val-Pro-Leu toward DP IV-catalyzed hydrolysis of Ala-Pro-pNA is (1.9±0.4)-10 -4 M (calculated as mean and standard deviation from all possibte intersections of the straight lines).
The data of Table II show that the value of k ~ , / K m of DP IV-catalyzed hydrolysis of tripeptides is generally lower than that for analogous dipeptide-4-nitroanifides. This is also true for the corresponding value of kcar In the case of tripeptides with an isoleucine residue in the Pl'-position, we found a remarkably low value of k~at and K m in comparison to other substrates. It is known that the rate.limiting step for the hydrolysis of substrates with a proline residue in the Pl-position is the deacylation reaction [7], which means that the k~t value is independent of the residue in the Pl'-position. This holds true, at least for substrates of the structure X-Pro-Y (X: amino acid; Y: arylamide). For natural substrates, where Y is any amino acid or peptide, we do not yet know the nature of the rate-limiting step of the hydrolysis of those compounds by dipeptidyi pcptidase IV. Considering the differences in the kea, values of the hydrolysis of tripeptid~ and dipeptide-4-nitroanilides, it may be suggested that with tripeptides the rate-limiting step of DP IV-catalyzed hydrolysis is at least partially the acylation reaction. Our results of the hydrolysis of tripeptides by dipeptidyl peptidase iV clearly show that those compounds without any doubt are substrates of DP IV. When the DP IV-catalyzed hydrolysis of a good substrate (e.g., Ala-Pro-pNA) is followed in the presence of a tripeptide (e.g., Val-Pro-Leu), the tripeptide leads to the picture of a kinetic competitive inhibition (Fig. 2). It can be shown by deriving the rate equation for the situation where one enzyme hydrolyzes two substrates
316 simultaneously that one substrate ($2) formally acts as the competitive inhibitor for the other substrate ($1) (e.g., SI: Aia-Pro-pNA; $2: Val-Pro-Leu). The Ki value in this case is not the true thermodynamic dissociation constant of the E-S2-complex but the K m value for the substrate $2, which is in the general case a complex kinetic constant. The g m value of the hydrolysis of Val-Pro-Leu by D P IV was estimated as (1.6 _+ 0.1)10 -5 M and the apparent K i value of the same compound towards the DP IV-catalyzed hydrolysis of AlaPro-plqA was (1.9 ± 0.4)-10 -5 M and in the case of Ala-AiaopNA as substrate (1.7 +_ 0.2). 10 -5 M, respectively. These values are identical and a support for the above mentioned suggestion. In conclusion it is appropriate to refer to diprotin A, diprotin B, as well as to other tripeptides with an analogous structure as substrates for dipeptidyl peptidase IV, An apparent competitive inhibition of DP IV
by those compounds is an interesting kinetic artifact which comes from the substrate-like nature of tripeptides with a penultimate proline residue.
References 1 Waher, R., Simmons. W.H. and Ynshimoto, T. (1980) Mol. Cell. Bio. 30. 111-127. 2 Heins. J. Welker, P., Sch~nlein. Chl. Born. L. Harlrodt. B.. Neubert, K~ Tsuru. D. and Barlh, A. (1988) Biochim. Biophys. Acta 954, 161-169. 3 Fischer, G. Heins, J. and Barth, A. (1983) Biochim. Biophys. Acta 742, 452-462. 4 Hein~ J, Neubet't, K., Scha~'weiB, B, and Barth, A. (1983) Federation Proceedings 42, 1324. 5 Kato, T~ Nagalsu, T., Fukasawa, K., Harada, M., Nagatsu, 1. and Sakakibant, S. (1978} Biochim. Biophys. Acta 525, 417-422, 6 Umezawa, H, Aoyagi. T., Ogawa, K., Naganawa, li., Hamada. M. and Takeuchi, T. (1984} J. Antibiotics 37, 422-425. 7 Wolf, B., F'~cl~r, G. and Barth, A. 0978) Acla Biol. Med. Germ. 37. 409-420.