A New Mechanism in Serine Proteases Catalysis Exhibited by Dipeptidyl Peptidase IV (DP IV) : Results...

Eur. .I. Biochem. 236, 109-114 (1996) 0 FEBS 1996

A new mechanism in serine proteases catalysis exhibited by dipeptidyl peptidase IV (DP IV) Results of PM3 semiempirical thermodynamic studies supported by experimental results

Wolfgang BRANDT', Olaf LUDWIG', Iris THONDORF' and Alfred BARTH'

' Department of Biochemistry/Biotechnology, Martin-Luther-University Halle-Wittenberg, Germany ' GBMC e.V. Halle, Germany

(Received 6 October 1995) - EJB 95 1637/4

Herein we present results of semiempirical molecular orbital calculations employing the PM3 molecular model. The compounds studied are related to substrates of the serine protease dipeptidyl peptidase IV (DP IV). Our goal was the thermodynamic characterization of the DP IV-enzyme-catalyzed reaction pathway. A new mechanism of serine proteases catalysis is presented. We found that a tetrahedral intermediate can be stabilized by the formation of an oxazolidine ring with the nonscissile P,-P, peptide bond. In this way, the negative charge of the tetrahedral intermediate around the scissile bond is transferred to the carbonyl oxygen atom of the preceding peptide bond. This negative charge can be compensated by a proton transfer from the positively charged N-terminus to this oxygen atom. It is shown that the positively charged N-terminus is the driving force in this particular serine protease mechanism of catalysis. The mechanism is supported by observed secondary hydrogen isotope effects on the C, proton for an alanine residue in the P, position. We suggest a truns-cis isomerisation around the P,-P, peptide bond in the final step of the acylation and cleavage of the substrates. The results obtained by our theoretical calculations are compared with several experimental findings supporting the suggested mechanism.

Keywords: dipeptidyl peptidase TV ; serine protease; catalysis mechanism; PM3 ; semiempirical calculations.

Dipeptidyl peptidase IV (DP IV) is a glycosylated dimer plasma membrane protein abundantly expressed on a variety of mammalian cells and tissues [l]. DP IV is a serine peptidase of broad medical and biochemical significance which plays a role in the degradation and post-translational processing of bioactive peptidcs such as substance P, P-casomorphins, growth-hormone- releasing factor and promelittin [2-91. DP 1V has been iden- tified as CD26, Tal, 1F7, Tp103 and the 110-kDa protein [lo- 141. It was demonstrated that it is a surface differentiation marker involved in the transduction of mitogenic signals in thy- mocytes and T lymphocytes in human, but recently also a cofac- tor in acquired immunodeficiency disease expression [ 10, 15 - 171.

The substrate specificity of DP IV has been well charac- terized. This enzyme sequentially removes a dipeptide from the N-terminus of a peptide chain whenever a proline residue or alanine residue is present in the penultimate position (scissile residuc, P, position using the nomenclature of Schechter and Berger [IS]) of the substrate. Peptides with proline at the P: position (C-terminal of the scissile residue) are not hydrolyzed. Any native amino acid can occupy the P, position (N-terminal from the scissile residue) as long as the N-terminus is unpro- tected and the N-terminal amino function is protonated. DP IV requires a truns peptide bond between the P, and P, residues

Correspondence to W. Brandt, Department of Biochemistry/Biotech- nology, Martin-Luther-University Halle-Wittenberg, Weinbergweg 16a, D-06099 Halle, Germany

1191.

Fax: +49 345 5527011. Abbreviations. DP IV, dipeptidyl peptidase IV ; TET, tetrahedral in-

Enzyme. DP IV. dipeptidyl peptidase IV (EC 3.4.14.5). termediate.

From kinetic investigations, it is known that deacylation is the rate-limiting step for alanyl-proline-4-nitroanilide at the pH optimum and the acylation reaction for alanyl-alanine-4-nitroanilide [20-221. There is no other serine protease known which requires the specific dipeptides (proline or alanine i n PI) and a positively charged N-terminus. It has, therefore, been suggested that this enzyme belongs to a new class of serine proteases [23, 241.

The role of the positively charged N-terminus is still not clear. Either, it may be important for the recognition of the substrates by forming a salt bridge with a negatively charged side chain of the enzyme, as suggested, or it is perhaps involved directly in the catalysis mechanism [25] ; both mechanisms simultaneously may also be possible.

In this paper, we present results which may help to clarify the situation. An alternative mechanism to the generally accepted catalytic pathway in serine proteases will be proposed for enzyme catalysis, probably exhibited by DP IV [26-281.

There are a multitude of theoretical investigations on the mechanism in serine protease catalysis (see for example, [29- 351). Most of them focus on the question of the validation of the so-called charge-relay mechanism, wich is the proton transfer from an active serine Oy to an imidazol ring and (questionable) simultaneous transfer of a proton from the imidazol to a side chain of an aspartic acid residue.

Other studies showed the main driving force in the enzyme catalysis to be the electrostatic field inside the active site of serine proteases [32]. It has been shown that the semiempiric PM3 method is more useful for studying enzyme catalysis than AM1 [36]. Therefore, we used this method for our investigation.

An X-ray crystal structure for DP IV does not exist. There- fore, we directed our attention only to thermodynamic consider-

110 Brandt et al. (Eur: J. Biochem. 236)

Scheme 1. The suggested reaction pathway of DPIV-catalysed substrate hydrolysis. Heats of formation are given in kJ/mol. Top : proton trarisfer from serine Oy to histidine as it usually occurs in serine proteases. Formation of the tetrahedral intermediate (TET,) by attack of the methanolate anion to the substrate 'Ala-Pro-NH(CH,) with fixed dihedral angle Y2 = 80". Chart: resulting geometry-optimized structure without a fixed dihedral angle Y2. The formation of an oxazolidine ring stabilizes the tetrahedral intermediate. Proton transfer from the positively charged N-terminiis to the negatively charged oxygen atom yields further energy gain, cleavage of the tetrahedral intermediate by proton transfer from the histidine cation to the nitrogen atom of the bond to be hydrolyzed and the resulting two alternative stable conformations of the formed dipeptide acylenzyme (ACE).

-217.3 88.8 -158.7 697.5

ED1 (-128.5) ED2 (538.8)

+ +H-Ala-Pro-NH(CH3) 247.4

CH-CH3 TETf

- 0-CH3 Q{:::;CH3 0 -154.1

o+c.'.-' CH-CH3 TETr

l CH-CH3 TETl -436.7 -282.6 '. 0 I 01

--.HNH2 NH3 { kH-CH3 TETl \ 01 '*-. HNH2 -436.7

CH-CH3 TET2 I

NH2 -578.6

697.5

CNLCf I 0-CH3

CNLCf O-.C+O N \ I O-CH3

C I 0-CH3

01

/C H3C-HC' *O 0' 'CH-CH~

NH3

ACEcis ACEtrans 56.1 74.1

+CH3NH* + N*N-H -21.8

ations of the reaction pathway of the catalysis mechanism. Although we developed a model for the active site of DP IV, arrangements of the probable catalytically active residues as a basis to estimate real activation barriers would be highly speculative 1251.

METHODS

All molecules were constructed with the molecular model- ling program SYBYL 6.la on an Evans and Sutherland workstation 10 and a Silicon Graphics Workstation VGXT (Tripos Associates Inc.). The calculations reported were carried out using the PM3 hamiltonian as implemented within the MOPAC 6.0 program in SYBYL [38]. The molecules were optimized

88.8

using the keywords PRECISE, MMOK, NLLSQ, FORCE and GNORM = 0.1.

All figures were prepared using the molecular graphics program for personal computer HAMOG and Word for Windows 6.0 [39].

RESULTS

Semiempirical molecular orbital calculations of the DP IV- catalyzed acylation step in the amide hydrolysis were performed (Scheme 1). The first step in serine proteases catalysis is assumed to be proton transfer from the serine Oy to histidine. We calculated the heats of formation for this process using methanol and methyl-imidazole to mimic the amino acid residues.

Brandt et al. (Eur: J. Biochern. 236) 111

The resulting reaction heat of formation was 667.4 kJ/mol, in correspondence with results obtained by Schroder et al. [36]. This very high positive heat of formation results from the calculation of the isolated molecules omitting stabilization by hydrogen bonds within the catalytic triad as well as the simultaneous formation of the tetrahedral intermediate. For a detailed analysis taking into consideration these aspects, see Daggett et al. [35].

As the first step in the hydrolyzation of the amide bond of DP IV substrates, the formation of a tetrahedral intermediate (TET; Scheme 1 ; Fig. 1 a) can be assumed, as usually accepted for serine proteases.

According to the substrate specificity of DP IV, we used H- Ala-Pro-NH(CH,) in its N-terminal protonated positively charged state as a model substrate. The starting conformation was taken from an earlier conformational analysis of substrates of DP IV [40]. Therein, a model was derived of the conformations probably adopted in the initial step (attack of the serine 0 7 ) of the catalysis by DP IV. We suggested a C, conformation ( Y, = SOo) around the P, residue (here proline) which could explain some aspects of the substrate specificity of DP IV and allows the attack of the serine Oy atom at the amide bond without steric hindrance.

The tetrahedral intermediate was formed by means of the SYBYL molecular graphics program package by placing a methanolate anion perpendicular to the scissile bond using the C7 starting conformation of the substrate. This structure was used as an input to a PM3 geometry optimization.

The resulting geometry-optimized structure yielded a surprising result at first glance (Fig. 1 b, TET,). An oxazolidine ring is formed by the attack of the oxyanion to the carbonyl carbon atom of the peptide bond preceding the proline residue. The formation of this intramolecular bond leads to a stabilization of the tetrahedral intermediate (AAH, = -525.4 kJ/mol) in an unusual manner. In this way, the negative charge of the oxyanion is transferred to the carbonyl oxygen atom of the first peptide bond (Scheme I). After this initial step, the negatively charged oxygen is in close proximity to the positively charged N-terminal nitrogen atom. For obvious reasons, one proton was transferred from the N-terminal nitrogen atom to the negatively charged oxygen atom. The resulting neutral compound (Fig. 1 c, TET,) is a further 141.9 kJ/mol more stable than the previous structure. Thus, the complete energy gain of this process is 667.4 kJ/mol, that is exactly the energy consumed for the proton transfer from a serine residue to a histidine residue. This is surprising when it is taken into consideration that no stabilization of the histidine cation by intermolecular hydrogen bonds was included in the calculations.

All serine proteases contain an oxyanion hole, which is generally formed by hydrogen-bonding donors (amide hydrogens) of the enzyme. It is widely accepted that the function of the oxyanion hole is to stabilize the tetrahedral intermediate by hydrogen bonding to the negatively charged oxygen atom. For comparison, these aspects were taken into consideration. For this purpose, the dihedral angle, Y2 = 80" was fixed in the starting conformation of ' H-Ala-Pro-NH(CH,) to prevent the formation of an oxazolidine ring and a restricted PM3 geometry optimization was carried out (Fig. 1 a, TET,). The heat of formation obtained for this structure (the usual tetrahedral intermediate) was only -282.6 kJ/mol in comparison to -436.7 kJ/mol for the oxazolidine structure or -578.6 kJ/mol after protonation of the oxyanion. The next question taken into consideration was: is this result an artifact of the semiempirical PM3 method? Such a mechanism has never been discussed for serine proteases. How- ever, there is no serine protease known which requires dipeptides with a positively charged N-terminus. Therefore, for com-

a

b

0

C

Fig. 1. Stereoviews of optimized structures along the most important steps in the suggested reaction pathway by DPIV substrate hydrolysis. (a) TET,; the dihedral angle !P2 = 80" has been fixed. (b) TET,, formation of the oxazolidine ring. (c) TET,, most stable conformation after proton transfer from the N-terminus to the particular oxyanion.

parison, the calculation was repeated using Acetyl-Ala-Pro- NH(CH,) as a model substrate for any other serine protease. The obtained fully optimized conformation was not as found above, but a tetrahedral intermediate as usually expected for common serine proteases. We forced a ring closure (oxazolidine ring) manually by SYBYL and performed another PM3 optimization. The resulting oxazolidine structure was only 7.5 kUmol more stable than the preceding one. This result and the high energy gain in the formation of the oxazolidine ring (154.1 kUmol or 296.0 kJ/mol, respectively) in the case of the DP IV substrate allows the conclusion to be made that the new mechanism is caused by the influence of the positively charged N-terminus inherent in the particular DP IV substrate structure and not by faults in the PM3 method. We assume that the probable substrate hydrolysis mechanism of DP IV is driven by electrostatic forces rising from the positively charged N-terminus. This electrostatic interaction attracts the first-formed negatively charged tetrahedral intermediate (TET,) on the scissile bond in a direction perpendicular to the imino bond and, in this way, the substrate sup- ports the enzyme catalysis by itself.

112 Brandt et al. (Eur: J. Biochem. 236)

Table 1. Heats of formation and reaction enthalpies of the enzyme- catalysis pathway of DP IV for H-Ala-Pro-NH[CH,]. AH, gives the sum of the heats of formation (additionally in ED, and ED,, AH, of the substrate ' H,-Ala-Pro-NH(CH,) = 247.4 kJ/mol) of all compounds regarded in Scheme 1. A,AH, indicates the reaction enthalpies between each step of the pathway, A,AH, indicates the reaction enthalpies taking into consideration the complete mechanism including the first step the proton transfer from Ser to His. Definition of lines head as described by Scheme 1. ED, educt; ACE, acylenzyme.

kJ/mol

ED 118.9 ED, 786.3 667.4 TET, 414.9 -371.4 296.0 TET, 260.8 -154.1 141.9 TET, 11 8.9 -141.9 0.0 ACE,,, 123.1 4.2 4.2

As the final step in this study, the formation of the acylenzyme by protonation of the nitrogen atom of the peptide bond to be hydrolyzed was considered. For this process, the proton transfer from histidine of the active site to this nitrogen atom can be assumed. It cannot, however, be excluded that the proton is transferred from the hydroxyl group in the tetrahedral intermediate (see Fig. I c , TET,) to the scissile bond and that, simultaneously, the N-terminal nitrogen atom is reprotonated by the solvent. However, independent from these two possibilities, the resulting formation of the acylenzyme structure can be obtained in a trans-proline or a cis-proline peptide bond. The result that is valid for both alternative structures of the finally formed acylenzyme is that the geometry optimizations immediately yield the cleavage of the peptide bond to be hydrolyzed (optimized C-N distance more than 180 pm). A conformation with a cis peptide bond between residues in the P, and P, positions is favorable by about 8.4-29.3 W m o l (see Tables 1 and 2). This is caused by interaction of the positively charged N-terminus with the car- bony1 oxygen atom of the ester group. Such a strong interaction is not possible in the case of a trans-proline peptide bond (Fig. 2). We assume a truns-cis isomerization to be possible since, in the formed particular tetrahedral intermediate, just this peptide bond is destabilized with regard to mcsomery stabilization. The carbonyl carbon as well as the nitrogen atom of the peptide bonds become sp' hybridized, as indicated by the bond angles (approximately 109") formed. A further indication is that the dihedral angle 0-C'-N-CG is no longer 180" but 110", which almost represents a transition state in a usual truns-cis isomer-

S P

'"g a

b

Fig. 2. Stereoview of the two alternative acylenzyme conformations (a) with a cis-proline peptide bond and (b) with a trans-proline peptide bond.

ization process. A trans-cis isomerization as a crucial step in acylation has already been proposed by Demuth [37].

The calculation was repeated for other substrates of DP IV and very similar results were obtained (Table 2).

DISCUSSION The above described new mechanism in serine protease cii-

talysis based on semiempirical calculations can be supported by the following experimental results. Barth [43] and Kiillerti et al. [44] investigated the substrate hydrolysis of H-Ala-Ala-4- nitroanilide by secondary hydrogen isotope effects concerning the P, position of this substrate. In analogy to the experimental conditions described in these experiments, we present here the results obtained for the H-D exchange of the proton of the Cu atom of the alanyl residue in the P, position (Heins, J. and Barth, A,, unpublished results; Fig. 3). From these results, a valuc of 0.85 f 0.09 for k,H,,/kFd, for the secondary hydrogen isotope et'fect concerning the P, position of 'H-Ala-Ala-4-nitroanilide has

Table 2. PM3 calculated heats of formation for some substrates of DP IV. MSC, most stable substrate conformation. The C-terminal group u5ed for all compounds was NH[CH,]. ACE, acylenzyme.

Compound Heat of formation for

kJ/mol

H- Ala-aziridide 332.9 -355.5 -508.3 144.9 170.4 H- Ala-oxazolidide 148.2 -560.2 -706.7 - 45.2 - 23.9 H- Ala-Pro 241.4 -436.7 -578.6 56.1 74.1 H- Ala-thiazolidide 344.2 -378.9 -505.8 157.4 180.5 H-Ala- flCS-N]-Pro 536.3 - 278.8 " -306.9h 356.3 365.9

Proton already linked to the sulphur atom. More stable conformation than in TET, with no hydrogen bonding between S-H and the N-terminal nitrogen atom.

Brandt et al. (EM J. Biochem. 236) 113

S/[Km + S]

Fig. 3. Observed secondary hydrogen isotope effect for the H-D exchange of the Ca proton in the P, position in the DP IV enzyme catalysis of H-Ala-Ala-4-nitoranilide.

been calculated. This result was not explainable, since this proton seemed to be relatively far from the reactive centre of the catalysis. It led to a number of speculations for a new enzyme hydrolysis mechanism occurring for DP IV [26-281. We can initially explain this effect using quantum chemical calculations. The formation of the oxazolidine ring in the catalysis brings the scissile bond close to the Ca proton at the P, position. This leads to differences in hyperconjugation effects and in steric interaction of the Ca proton in the P, position with the carbonyl oxygen atom of the peptide bond between P, and P, positions during the enzyme catalysis. These might be the reasons for the observed effect. The experimental result clearly shows that the amino acid residue in the P, position takes part in the rate-limiting step of the enzyme catalysis and, therefore, may be a strong indication for the suggested reaction path way.

In a recently published study, the synthesis and kinetic results of a thioxoalanyl-proline-4-nitroanilide DP IV substrate are described [46]. The substitution of the carbonyl oxygen atom by a sulphur reduced the K,,, and the k,,, by about three magnitudes [46]. The very high decrease in these constants seems to be un- explainable on the basis of a reduced binding affinity (Michaelis complex) for the enzyme alone. These results may serve as a further experimental indication of a direct participation of the thioxo group in the catalysis mechanism as described above. Schutkowski et al. [46] determined experimentally a higher free energy of activation in the enzyme catalysis for this compound than for the usual Ala-Pro analogue. They discussed a cis-trans isomerisation of the H-Ala- flCS-NI-Pro-4-nitroanilide to be the rate-limiting step and, therefore, to be responsible for the low- ered k,,, and higher free energy of activation, respectively. In our opinion, the reversed process (trans-cis instead of cis-trans isomerisation), as discussed above, would also explain these experimental facts. It is known that DP IV requires trans conformations for substrates or inhibitors [19]. The dipeptides are known to be effective inhibitors for DP IV. However, if the formation of a cis-proline peptide bond in the catalysis mechanism is assumed, the resulting dipeptides will be removed very fast

from the binding pocket and will not inhibit the enzyme, due to a low binding affinity. An indication for the occurrence of a trans-cis isomerisation is given more indirectly by several studies. Sudmeier et al. [47] investigated boronic acid substrates at different pH values by means of NMR spectroscopy. They demonstrated the formation of a cis-proline peptide bond at neutral pH, together with the formation of a ring structure formed between the N-terminus and C-terminus. The formation of di- ketopiperazines is also often observed as side reactions in the dipeptide synthesis of DP IV substrates that includes a cis peptide bond. It is assumed that the deacylation of proline (in P,) substrates is the rate-limiting step of enzyme catalysis by DP IV. This would be in contradiction to our hypothesis of a trans-cis isomerisation in the acylation process to be rate limiting. Either the trans-cis isomerisation occurs in the case of proline substrates in the deacylation step in a similar way as described here, or the interpretation of a number of experimental results has to be reconsidered. The conclusion that deacylation is rate limiting in proline substrates has been drawn from influences of solvent effects to the kinetic constants [22,46] as well as from quantita- tive structure activity analysis studies [20, 211. There is no doubt that a solvent is able to influence the state of protonation of the N-terminal nitrogen atom and, therefore, the proton transfer to the carbonyl oxygen atom (TET, to TET, or, alternatively, a reprotonation of the N-terminal nitrogen atom; see above). This process leads to the essential stabilization of the tetrahedral intermediate (switch from an endothermic reaction to an exothermic reaction; Table 1) and should be influenced by the type of solvents or by the pH value. If this reaction is related to the rate- limiting step together with the synchronous trans-cis isomerization, then the solvent effects would also be explainable in the acylation process. This is supported by the finding that, at pH 5.6 and lower, the rate-limiting step, even in the proline substrates, becomes the acylation reaction. Moreover, solvent isotope studies carried out by Kaiser [45] showed that, for proline in the P, position (at pH 7 . 3 , one proton is involved in the rate- limiting step of the enzyme catalysed by DP IV, but for alanine substrates, two protons are involved. From these results, one can conclude that, in the case of proline substrates, the one proton transfer, as described above, is an essential part of the rate-limiting step, whereas in the case of alanine substrates, both this proton transfer together with the protonation of the scissile bond may determine the rate of the acylation reaction. The barrier against the necessary rotation in the trans-cis isomerisation is no longer determined by the mesomery stabilization of the peptide bond, but by the steric strain of the side chain of the amino acid residue in the P, position, with the CG-methylene group of the proline substrates which is not existent in alanine substrates. Thus, the rotation barrier should be lower in the alanine substrates than in the proline substrates, which would explain the suggestions above. Calculations of this rotation will be carried out later using ab initio methods, since it was shown that semiempirical methods are inadequate for the calculation [48]. QSAR analyses were carried out on the basis of different anilide derivatives substituted by electron-withdrawing as well as electron-delivering substituents in both the proline and alanine substrates. In the case of alanine substrates, a correlation of k,,, with hammett substitution constants was found, but for proline substrates no correlation was found [20, 211. This led to the conclusion that deacylation is the rate-limiting step in proline substrates. However, if indeed the considered proton transfer and the trans-cis rotation of the peptide bond are correlated with the rate-limiting step in the proline substrates, no correlation or only very weak correlations are found.

Thus, all the considered experimental results that interpret deacylation as rate limiting in proline substrates are not conclu-

114 Brandt et al. (Eul: J. Biochem. 236)

sive enough to exclude acylation as the rate-limiting step for this class of substrates.

D P IV is difficult to inhibit with diisopropylfluorophosphate or phenylmethanesulfonofluorate [49]. These difficulties may be an indication for both an incomplete catalytic triad in DP IV and for the above described mechanism in enzyme catalysis. These inhibitors are not able to form such a particular tetrahedral intermediate discussed and cannot easily react with the active serine residue if the catalytic triad is incomplete. Another explanation, however, is discussed in [25]. Usually enzyme catalysis in serine proteases has to be supported by stabilization of the imidazole cation by the aspartic acid anion of the catalytic triad. In the case of DP IV, this seems not to be absolutely necessary, since the described reaction pathway becomes exothermic without stabilization by intermolecular hydrogen bonds. Mutation experiments showed the importance of an aspartic acid residue in the catalysis of D P IV [SO, 511. However, one can assume that the positively charged N-terminus will be recognized in the formation of the Michaelis complex by a negatively charged side chain, that may be this aspartic acid, too. We were able to de- velop a model of the active site of DP IV that exactly shows this recognition [25].

The results presented in this paper offer not only a new mechanism in serine proteases valid for D P IV, but may explain some experimental facts (Fig. 3) based on semiempirical PM3 calculations and lead to new interpretations of a number of experimental observations (Heins, J. and Barth, A,, unpublished results). Nevertheless, it is obvious that this mechanism has to be proven by further experimental as well as theoretical investigations.

We are grateful for research support (grant 1185A/0083) from the Miriisterium f u r Wissenschaji und Forschung von Sachsen-Anhalt (Ger- many) given to I. T. and W. B.

REFERENCES 1.

2.

3. 4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

3 4.

15.

16. 17.

Hartel, S . , Gossrau, R., Hanski, C. & Reutter, W. (1988) Hisfochem.

Nausch, I., Mentlein, R. & Heymann, E. (1990) Biol. Chern. Hoppe-

Nausch, I. & Heymann, E. J. (1985) Neurochem. 44, 1354-1357. Ahmad, S . , Wang, L. &Ward, P. E. J. (1992) Pharmacol. Exp. Thel:

Hartrodt, B., Fischer, G., Schulz, H. & Barth, A. (1982) Pharmazie

Kikuchi, M., Fukuyama, K. & Epstein, W. L. (1988) Arch. Riochrm.

Kreil, G., Umbach, M., Brantl, V. & Teschemacher, H. (1983) Life

Frohman, L. A,, Downs, T. R., Heimer, E. P. & Felix, A. M. (1989)

Kreil, G., Haiml, L. & Suchanek, G. (1980) EUI: J. Biochem. 111,

Callebaut, C., Krust, B., Jacotot, E. & Hovanessian, A. G. (1993)

Ulmer, A. J., Mattem, T. & Flad, H.-D. (1992) Scand. J. Immunol.

Hafler, D. A,, Choflon, M., Benjamin, D., Dang, N. H. & Breit-

Hegen, M., Camerini, D. & Fleischer, B. (1993) Cell Immunol. 146,

Kreisel, W., Volk, B. A., Buchsel, R. & Reuter, W. (1980) Proc. Natl Acad. Sci. USA 77, 1828-1831.

Fox, D. A., Hussey, R. E., Fitzgerald, K. A,, Acuto, O., Poole, C., Palley, L., Daley, J. F., Schlossman, S . F. & Reinherz, E. L. J. (1984) Immunol. 133, 1250-1256.

89, 151-161.

Seyler 371, 1113-1118.

260, 1257-1256.

37, 165-169.

Biophys. 266, 369-376.

Sci. 33, 137-140.

J. Clin. Invest. 83, 1533-1540.

49-58.

Science 262, 2045 -2050.

35,551-559.

meyer, J. J. (1989) Immunol. 142, 2590-2596.

449 -460.

Fleischer, B. J . (1987) Immunol. 1-38, 1346-1350. Reinhold, D., Bank, U., Buhling, F., Neubert, K., Mattem, T., Ulmer,

A. J., Flad, H.-D. & Ansorge, S . (1993) lmmunohiol. 188,403-414.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38. 39.

40.

41.

42.

43. 44.

45.

46.

47.

48.

49.

50.

51.

Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Comriiun. 27, 157-162.

Fischer, G., Heins, J. & Barth, A. (1983) Biochim. Biophys. \cfa 742, 452-462.

Fischer, G. & Barth, A. (1981) in Beitriige zur Wirkstoflor~scllurig (Ohme, P., Lowe, H. & Gores, E., cds) vol. 11, pp. 105- 133, Akademie-Industrie-Komplex Arzneimittelforschung, Berlin.

Barth, A,, Fischer, G., Neubert, K., Heins, 5. & Mager, H. (1080) Wiss. Z. Leuna-Merseburg 22, 352-371.

Rahfeld, J., Schutkowski, M., Faust, J., Neubert, K., Barth, A. & Heins, J. (1991) J. Biol. Chem. Hoppe-Seyler 372, 313-318.

David, F., Bernard, A.-M., Pierres, M. & Marguet, D. J. (1 993) Riol. Chem. 268, 17247-17252.

Polgar, L. & Szabo, E. J. (1992) R i d . Chem. Hoppe-Seyler 373, 361 -366.

Brandt, W., Lehmann, T., Thondorf, I., Born, I., Schutkowski. M., Rahfeld, J., Neubert, K. & Barth, A. (1995) Int. J. Peptide Protein Rex 46, 494-507.

Barth, A,, Wahab, M., Brandt, W. & Frost, K. (1993) Drug Design Discovery 10, 297 - 317.

Barth, A,, Brandt, W., Wahab, M., Frost, K., Schadler, H.-I). & Franke, R. (1994) Drug Design Discovery 12, 89-111.

Stein, R. L., Fujihara, H., Quinn, D. M., Fischer, G., Kullertc. G., Barth, A. & Schowen, R. L. (1984) J. Am. Chern. Soc. 106,

Warshel, A. & Russell, S . (1986) J . Am. Chem. Soc. 108, 6269-

Scheiner, S . , Kleier, D. A. & Lipscomb, W. N. (1976) Proc. Natl

Dewar, M. J. S . & Storch, D. M. (1985) Pmc. Natl Acad. Sci. USA

Warshel, A. (1991) Computer modeling c$ chemical reaction 1 1 1 rt1- zymes and solutions, pp. 170-188, John Wiley & Sons, Inc. New York, Chichester, Brisbane, Toronto, Singapore.

Umeyama, H., Imamura, A., Nagato, C. & Hanano, M. J. (1973) Theol: B i d . 41, 485.

Hayes, D. M. & Kollman, P. A. (1979) in Catalysis in cheniisrrj and hiochernistry (Pullman, B., ed.) D. Reidel Publishing Co., Boston, MA.

Daggett, V., Schroder, S. & Kollman, P. (1991) .I. Am. Chenl. Soc.

Schroder, S., Daggett, V. & Kollman, P. (1991) J . Am. Chem Soc.

Demuth, H.-U. (1988) Doctoral Thesis, Martin-Luther-University.

Stewart, J. J . P. (1989) J. Cornput. Chem. 10, 209-220. Brandt, W., Wahab, M., Thondorf, I., Schinke, H. & Barth, A.

Brandt, W., Lehmann, T., Hofmann. T., Schowen, R. L. & Barth. A.

Harada, M., Fukasawa, K., Hiraoka, B. Y., Mogi, M., Barth. A. &

Berger, E., Fischer, G., Kullertz, G. & Barth, A. (1987) Biomed.

Barth, A. (1979) Wiss. Z. Univ. Halle XXVIII’79M 3. 23-48. Kullertz, G., Fischer, G. & Barth, A. (1976) Trtrahedw? 32,

Kaiser, M. (1985) Ph.D. Thesis Martin-Luther-University, Halle-

Schutkowski, M., Neubert, K. & Fischer, G. (1994) Eul: J. Biochrm.

Sudmeier, J. L., Gunther, U. L., Gutheil, W. G., Coutts, S . J.. Snon., R. J., Barton, R. W. & Bachovchin, W. W. (1994) Biochetnisfr),

Feigel, M. & Strassner, T. J. (1993) Mol. Stvucf. Theoclien~. 283,

Barth, A,, Schulz, H. & Neubert, K. (1974) Acta Biol. Mrd. Ger.

Ogata, S., Misumi, Y., Tsuji, E., Takami, N., Oda, K. & Ikehara, Y.

Misumi, Y., Hayashi, Y., Arakawa, F. & Ikehara. Y. (1992) Bicichim.

1457-1461.

6579.

Acad. Sci. USA 73, 432.

82, 2225-2229.

113, 8926-8935.

113, 8922-8925.

Halle-Wittenberg, 35-60.

(1991) J. Mol. Graphics 9, 122-126.

(1992) J. Cornput.-Aided Mol. Des. 6, 159-174.

Neubert, K. (1985) Biochim. Biophys. Acfa 830, 341-344

Biochim. Acta 46, 671 -676.

759-761.

Wittenberg, Germany.

221, 455-461.

33, 12 427- 12438.

33-48.

32, 157-174.

(1992) Biochemistry 31, 258252587,

Riophys. Acfa 1131, 333-336.

A New Mechanism in Serine Proteases Catalysis Exhibited by Dipeptidyl Peptidase IV (DP IV) : Results...

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