Vol. 187, No. 2, 1992

September 16, 1992

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 615-619

EFFECT OF P2’ SUBSTITUENTS ON KINETIC CONSTANTS FOR HYDROLYSIS BY CYSTEINE PROTEINASES

Carlos Garcia-Echevetia, and Daniel H. Rich

School of Pharmacy and Department of Chemistry, Universig of Wisonsin-Madison 425 N Charter St., Madison, WI 53706

Received July 24, 1992

Summary: Intramolecularly quenched fluorogenic peptide substrates with the general sequence: DABCYL-Lys-Phe- Gly-Gly-Xxx-Ala-EDANS have been utilized to explore the effect of the hydrophobicity of amino acid side chains in the P2’ position on the steady-state kinetic constants for papain catalyzed hydrolysis. The results demonstrate that subsite interactions between the enzyme and the peptide substrate modulate the enzyme specificity by slowing the release of the C-terminal product. This series of substrates can be used to characterize substrate specificity studies of other cysteine proteinases. 0 1992 Academic Press, Inc.

Cysteine proteinases (EC.3.4.22) constitute a group of structurally and functionally related

proteins, which owe their enzymatic activity to the presence of a thiol group in the active site (l-3).

Members of this class of enzyme are widespread in Nature and include plant proteinases: papain,

ficin, bromelain, and actinidin; mammalian cytoplasmic Ca 2+-dependent enzymes: calpain I and II;

and mammalian lysosomal enzymes: cathepsin B, H, S, and L. Many pathological conditions (4-

12) such as muscular dystrophy, amyloidosis, inflammatory and heart diseases, or tumor

invasiveness are believed to be initiated and/or sustained by some of the above endopeptidades. A

better understanding of the catalytic mechanism and specificity of these proteinases is thus of

interest to facilitate the design and development of specific, non-toxic cysteine proteinases

inhibitors (12,13), which could potentially be developed into therapeutic drugs.

The high homology among cysteine proteinases allows the information obtained from one

enzyme to be used to understand the mode of action and specificity of related enzymes. Such an

approach has been used with papain (EC.3.4.22.3), a cysteine proteinase isolated from the latex of

papaya, which is taken as the prototypic member of this class of enzymes. Even though the

catalytic mechanism of papain has been characterized (14), a limited number of structure-activity

studies of the prime region of the enzyme are available (15-18). We have shown (18) that the use

of intramolecularly quenched fluorogenic peptide substrates provides a method to obtain kinetic

Abbreviations: Boc, terr-butyloxycarbonyl; DABCYL, 4-(4-dimethylaminophenylazo)benzoic acid; DMSO, diiethylsulfoxide; E-64, trans-epoxysuccinyl-L-leucylamido@-guanidino)butane; FDTA, ethylenediaminetetraacetk acid; EDANS, 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid; FABMS, fast atom bombardment mass spectrometry; HPLC, high pressure liquid chromatography; MCA, methylcoumarylamide; TFA, trifluoroacetic acid; tR, retcudou the; z, benzyloXycarbouy1.

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Vol. 187, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

data in the prime region of cysteine proteinases, which was not attainable with chromophoric

substrates previously employed to characterize these enzymes. Kinetic analyses are based on

resonance energy transfer (19-22) between the DABCYL (quencher) and EDANS (fluorophore)

groups (23), which permits one to monitor the appearance of the product, H-Gly-Xxx-Ala-

EDANS. In the present paper, we evaluate the effect of amino acid hydrophobicity in the P2’

position [notation of Schechter and Berger (15)] on the kinetic parameters for papain-catalyzed

hydrolysis of DABCYL-Lys-Phe-Gly-Gly-Xxx-Ala-EDANS (where Xxx= Phe, Leu, Val, Ala,

Asn). The results suggest that C-terminal product release is an important contributor to the minimal

catalytic mechanism for hydrolysis of peptide substrates.

Materials and Methods

Papain and E-64 were purchased from Sigma and used without further purification. The substrate Na-Z-Phe- Arg-MCA, and all Boc-amino acids were purchased from Bachem Inc.

The lluorogenic peptidyl substrates were synthesized by classical solution methods using procedures reported elsewhere (lg), and purified by reverse-phase high pressure liquid chromatography on a Waters Bondapak C-18 column (7.8 x 300 mm). Final products were evaluated by analytical HPLC on a Vydac C-18 column (4.6 x 250 mm), linear gradient over 20 min of CHjCN-0.036 % TFA and H20-0.045 % TFA from I:19 to 4: 1, flow rate 1.2 mL/min, detection at 254 nm; tR= 16.7 min (a); 16.5 min (b); 16.1 min (c); 16.1 min (d); 15.8 min (e). The integrity of each purified peptide was determined by FABMS, and the observed molecular mass was found to agree with the calculated values.

Fluorescence was monitored by using a Perkin-Elmer fluorescence spectrometer model MPF-4 equipped with a Xenon Power Supply 156, and a recorder model 56. Papain activity was assayed with Na-Z-Phe-Arg-MCA at pH 6.45 according to the method of Barrett et al. (24). The fluorescence of the 7-amine-4-methylcoumarin group liberated from the substrate was monitored at 458 nm with excitation at 370 nm. The active concentration of papain was determined by active site titration with E-64 for 30 min at 30 oC as described (24).

Hydrolyses by papain of the above ffuorogenic substrates were monitored at 30 OC in 1 cm cuvettes. The poorly soluble substrates were dissolved in DMSO and diluted with buffer; final concentration of DMSO was = 2 %. All reactions were conducted in 0.4 M phosphate buffer (pH= 6.45) containing 8 mM dithiothreitol, and 4 mM EDTA. The excitation wavelength was 336 nm and the emission wavelength was 472 nm with a slit of 10 nm. Initial velocities’ data for determining the steady-state kinetic constants were obtained in triplicate by continuously recording the fluorescence for 5 min. For each substrate in Table 1, the Michaelis-Menten constant (KM) and the turnover number (k& with respective standard errors were evaluated through the Michaelis-Menten equation using the nonlinear least-squares method &larquardt algorithm (25)]. All peptides were cleaved at one site, which was identified by analytical HPLC of the products.

Results

Hydrolysis of five substrates, DABCYL-Lys-Phe-Gly-Gly-Xxx-Ala-EDANS (Xxx is Phe

(a), Leu (h), Val (c), Ala (d), and Asn (e)), by papain was accompanied by an increase in the

fluorescence intensity due to a release of the quenching of the fluorophore. The increase in

fluorescence was proportional to the concentration of the peptide fragment H-Gly-Xxx-Ala-

EDANS, which was identified for each substrate by analytical HPLC after exhaustive enzymic

digestion. The fluorescence of this peptide moiety was used as a reference in the analysis of the

fluorescence spectra recorded during the enzymatic digestion.

Table 1 shows the steady-state kinetic constants for the hydrolysis of a-e. Partition coefficients

(1ogP) for each amino acid side chain in the P2’ position were calculated from atomic increments

by using the values reported by Ghose & Crippen (26), and were employed to evaluate the effect

of the hydrophobicity on the kinetic parameters of the corresponding substrate.

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Vol. 187, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1 Steady-state kinetic constants for papaina catalyzed hydrolysis of intramolecularly quenched

fluorogenic substrates a-e

H

Lys-Phe-Gly-@Xxx-Ala /NWNH

Substrate P2 ’ (2)

~xJKM (M-’ s-1)

logpb

a Phe 0.44 + 0.07 1.01 * 0.28 x 10-5 4.30 f 0.58 x 104 2.383

b kU 1.61 f 0.41 2.18 f 0.96 x 10-5 7.41 + 1.48 x 104 1.932

C Val 2.47 f 0.59 1.58 +I 0.67 x 10-5 1.57 f 0.30 x 105 1.470

d Ala 3.30 f 0.30 0.76 + 0.21 x 10-5 4.33 + 1.28 x 105 0.659 e Asn 1.14 + 0.14 1.41 f 0.50 x 10-5 8.07 f 1.55 x 104 - 0.858

a Kinetic constants were determined as described in Materials and Methods. b Values taken from reference (26).

Discussion

Most chromogenic peptide substrates have been designed to liberate the chromophoric group

upon hydrolysis from the cleavage site, which precludes kinetic studies to qua&ate the effect of

interactions between the enzyme and the prime regions of biologically important substrates, e.g.

peptides or proteins. Recently, we have shown (18) that systematic kinetic studies of the

interactions of cysteine proteinases with the prime region of substrates can be carried out by using

a continuous fluorimetric assay based on resonance energy transfer (19-22). In this assay, the

spatial proximity between the quencher (DABCYL) and the fluorophore (EDANS) linked by a

peptide causes a reduction in the fluorescence of the EDANS group; proteolytic cleavage of the

connecting peptide substrate releases the quenching with a fluorescence quantum yield proportional

to the concentration of the fluorophote fragment (18,23).

Our results show that single amino acid replacements in the P2’ position of the substrate

DABCYL-Lys-Phe-Gly-Gly-Xxx-Ala-EDANS effect the steady-state kinetic constants (27) for

papain catalyzed hydrolysis (Table 1). Figure 1 shows the correlations obtained between the

primary kinetic constants, kcat and bat/KM and the logarithm of the water-octanol partition

coefficient (logP), which expresses the hydrophobicity of the Xxx-amino acid side chain.

Although only a limited number of analogues have been synthesized and evaluated, the

relationships between kat/KM and bat and hydrophobicity appear to be parabolic. The highest

value for the specificity constant k&KM was obtained when Xxx= Ala; replacement of this amino

acid by Asn,, Val, Leu or Phe decreased &at/KM 2 to lo-fold. A similar trend was observed for the

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Vol. 187, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

“-1 0 1 2 3

log P

-1 0 1 2 3

log P

Figure 1 Correlation between the k,-at and ~&KM values and 1ogP for the P2’ position of the

intramolecularly quenched fluorogenic peptide substrates: DABCYL-Lys-Phe-Gly-Gly-Xxx-Ala-EDANS (Xxx: Phe, Leu, Val, Ala, Asn).

turnover number (keat). Since both keat and keat/K~ are affected similarily, only those kinetic

steps common to kcat and bat/KM are likely to be affected by log P. This restricts the kinetically

observed effects of hydrophobicity reported here to steps that occur&&r the Michaelis complex is

formed, but before the first irreversible step, which is release of H-Gly-Xxx-Ala-EDANS (27).

The decrease in kinetic constants with 1ogP greater than 0.5 is consistent with increased

hydrophobic interactions between the C-terminal fragment (H-Gly-Xxx-Ala-EDANS; Ala, Val,

Leu, Phe) and the S2’ enzyme binding site that retard the release of the C-terminal product. The

increase in kcat going from substrate with Xxx=Asn to Xxx=Ala is consistent with an enhanced

acylation step due to tighter binding of substrate P2’ substituents in the transition state for this step

(28). The effects of hydrophobic interactions on kinetic constants are likely to be difficult to detect

because of their opposite effects depending on which step in the mechanism is more affected.

Cysteine proteinases hydrolyze amide and ester bonds by a mechanism involving at least

four-steps: i) substrate binding; ii) acylation of the cysteine residue in the active site; iii) release of

the first product P2; and iv) hydrolysis of the acylenzyme intermediate (deacylation) with release of

the second product Pt. In previous studies with chromogenic substrates, the invariant nature of the

chromophoric group (P2 product) and its rapid release simplified the mechanism to steps i, ii and

iv. The present study establishes that in substrates more closely related to natural substrates,

hydrophobic interactions between enzyme and C-terminal portion of substrate affect post acylation

steps. The decrease in k,t and kcat/KM is consistent with increased barriers to P2 release. The

effect observed for P2’ substituents differs from that observed for KM or k&KM when the PI’

position was evaluated (18). It is likely that specificity toward endogenous protein substrates may

also be affected by product release steps, in addition to specificity contributions from binding and

acylation steps (16).

AcknmvZe&ments: We thank Dr. Petr KuzmiE for the use of his FastKi program and Mr. Z.-C. Zhao for his technical assistance. The provision of a NATO post-doctoral fellowship to C. Garcfa- Echeverrfa and the support from the National Institutes of Health (GM 40092) are gratefully acknowledged. Mass spectra were performed by the Midwest Center for Mass Spectrometry and NSF regional instrumentation facility (CHE 8620177).

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:: 3.

4. 5.

6.

7.

;:

10. 11.

12.

13 14.

15. 16. 17.

18.

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2. 23:

24.

25. 26. 27.

28.

References

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