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Biochemistry 1992, 31, 126 18-1 2623
Substrate Specificity of the Human Matrix Metalloproteinase Stromelysin and the Development of Continuous Fluorometric Assays Lisa Niedzwiecki, Jennifer Teahan, Richard K. Harrison, and Ross L. Stein’J Department of Enzymology, Merck & Co., P.O. Box 2000, Rahway, New Jersey 07065 Received August 18, 1992; Revised Manuscript Received October 7 , I992
ABSTRACT: To probe the specificity of the metalloendoproteinase stromelysin toward peptide substrates, we determined k , / K , values for the stromelysin-catalyzed hydrolyses of peptides whose design was based loosely on the structure of a known S L N substrate, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-GlyLeu-MetNH2, hydrolysis a t Gln-Phe, k , / K , = 1700 M-I s-l). Several noteworthy points emerge from this study: (i) Catalytic efficiency is dependent on peptide chain length with N-terminal truncation of substance P resulting in more pronounced rate-constant reductions than C-terminal truncation. These results suggest the existence of an extended active site for stromelysin. (ii) Preferences a t positions P3, P2, P I , PI’, and P2’are for the hydrophobic amino acids Pro, Leu, Ala, Nva, and Trp, respectively. (iii) Investigation of specificity a t P3’supports our earlier hypothesis that S L N has a requirement for a hydrogen-bond donor at this position in its substrates. Based on these observations, we designed and had synthesized the fluorogenic substrate N-(2,4-dinitrophenyl)Arg-Pro-Lys-Pro-Leu-Ala-Nva-TrpNH~, whose stromelysin-catalyzed hydrolysis can be monitored continuously ( k , / K , = 45 000 M-’ s s ‘ ) .
Stromelysin is a member of the matrix metalloproteinase family (Emonard & Grimaud, 1990; Matrisian, 1990) and is thought to play a pathogenic role in arthritis (Emonard & Grimaud, 1990; Hasty et al., 1990; Matrisian, 1990). As part of a program to develop therapeutic inhibitors of this and other MMPs,l we are studying mechanistic aspects of metalloproteinase catalysis and inhibition (Izquierdo & Stein, 1990; Izquierdo-Martin & Stein, 1992a-c; Stein, 1988). An important mechanistic issue is the definition of SLN’s substrate specificity. An earlier study (Teahan et al., 1989) from our laboratory reported data on the SLN-catalyzed hydrolyses of various analogues of substance P, Arg-Pro-LysPro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2. SLN cleaves this peptide exclusively at the Gln6-Phe7bond with kinetics that allowed the development of a semicontinuous, HPLC-based assay (Harrison et al., 1989). In this paper, we continue our exploration of the substrate specificity of SLN with the two goals of defining substrate structural requirements for efficient catalysis by SLN and laying the foundation for the development of a continuous assay for this enzyme. To determine SLN’s requirements for its substrates, we measured values of k,/K, for SLN-catalyzed hydrolyses of peptides that were designed to probe the extended active site of SLN as well as subsites S3 through S3’.2 For the second goal of developing a continuous assay, our strategy was to use the specificity results to design an optimal peptide substrate for SLN and then incorporate this structure into a suitable format for continuous assay. Our studies led us to the optimal peptide, Arg-Pro-Lys-Pro-Leu-Ala-NvaTrpNH2, and its simple fluorogenic derivative DNP-Arg-
* To whom correspondence should be addressed.
* Current address:
Genesis Pharmaceuticals, 840 Memorial Dr., Cambridge, MA 02139. I Abbreviations: MMP, matrix metalloproteinase; SLN, stromelysin; CGase, collagenase; TLN, thermolysin; DNP, 2,4-dinitrophenyl; Ac, acetyl; Nva, norvaline; Abu, a-aminobutyric acid; SP, substance P. * The nomenclature for the amino acid residues of the substrate ( P,- ...-P~-P~-PI-PI’-P~’-P~’....Pn) and the corresponding protease subsites (S,S~-S~-SI-SI’-S*’-S~’S ~is) that of Schecter and Berger (1967). Hydrolysis occurs at the Pl-PI’ bond.
0006-2960/92/0431-12618$.03.00/0
Pro-Lys-Pro-Leu-Ala-Nva-TrpNH2, whose hydrolysis can be followed continuously (Netzel-Arnett et al., 1991b; Stack & Gray, 1989).
MATERIALS AND METHODS Materials. Peptide substrates were purchased from Multiple Peptide Systems (San Diego, CA) or Bachem (Philadelphia, PA) at a purity greater than 95%. Recombinant human prostromelysin (Whitman et al., 1986) was purchased from Celltech Ltd. (Slough, Berkshire, U.K.) at a concentration of 100 pg/mL and a purity of 98% and in a buffer of 25 mM Tris-HC1, 10 mM CaC12, 0.1% sodium azide, and 0.05% Brij-35, pH 7.5. Trypsin-catalyzed activation of proSLN was according to published procedures (Teahan et al., 1989; Lark et al., 1990a,b). Substrate specificity assays were performed in a buffer of 0.1 M HEPES and 0.01 M CaC12, pH 7.5. Kinetic Experiments-Semicontinuous, HPLC-Based Assay. The general features of this assay have been described previously (Harrison et al., 1989; Teahan et al., 1989). The chromatographic aparatus consisted of a Waters 5 10 HPLC System (Division of Millipore, Millford, MA) and a Hitachi model 655A-40 Autosampler fitted with a temperaturecontrol option ( E M . Science, Cherry Hill, NJ). Temperature was maintained at 25.0 f 0.1 “ C by a Lauda RM6 refrigerated circulating water bath (Brinkmann, Westbury, NY). UV absorbance was measured at 215 nm using a Kratos Spectroflow 757 variable wavelength detector (AB1 Analytical, Ramsey, NJ). Fluorescence emission was measured with an Hitachi F1200 fluorescence detector at A,, 280 nm and A,, 345 nm. Peak integrations were performed on a PE Nelson Turbochrom Data Station (Cupertino, CA). Separations were performed on a Whatman Partisil5 C8 analytical column (25 cm X 4.6 mm i.d.). The mobile phase was 0.1% TFA aqueous and CH3CN. The flow rate was 1.5 mL/min. Determination of k,/K,,, Values. In a typical kinetic measurement, 0.90 mL of a buffered solution of substrate in a 2.0-mL glass autosampler vial was thermally equilibrated to 25 ‘C for 15 min in the thermostated compartment of the
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Biochemistry, Vol. 31, No. 50, 1992 12619
Stromelysin Specificity autosampler. A 0.90-mL aliquot of a diluted sample of [SLNIst,t was then added to this vial to give final SLN and substrate concentrations of 0.035 and 50 pM, respectively. The reaction solution was mixed by inversion and placed back in the autosampler. Immediately after the initiation of the reaction, and at predetermined time intervals thereafter, 20pL aliquots were withdrawn from the reaction solution by the autosampler and injected onto the column. Loss of substrate was quantitated either by absorbance at 215 nm or by tryptophan fluorescence. Values of k c / K , were calculated according to the expression k,/K, = k,b,/[E], where kobs is the observed first-order rate constant for loss of substrate obtained from progress curves recorded under the condition that [SI, = 50 pM
