Stromelysin

Stromelysin, which degrades proteoglycan and other proteins of the matrix of cartilage, is a metalloendoproteinase secreted by mammalian fibroblasts. The assay described here uses substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Gly-Leu-Met-NH2) in an automated, semicontinuous assay that is well suited for kinetic analyses. Cleavage occurs specifically between positions 6 and 7 of the peptide. 

Stromelysin was isolated as the zymogen from the culture media of interleukin-1-stimulated human gingival fibroblasts. The zymogen is activated by incubating with trypsin, which in turn is inactivated by adding soybean trypsin inhibitor. 

Dipeptidyl peptidase IV hydrolyzes Xaa-Pro-Yaa- peptides to Xaa-Pro and Yaa-, while amino peptidase-P cleaves on the amino side of Pro. These two enzymes are found in the microvillous fraction of rabbit kidney. Catalysis is nonspecific with respect to Xaa and Yaa, except the Yaa position cannot be occupied by Pro or Hyp. Harada et al. have published several papers on assaying cleavage of proline-containing peptides found in structural proteins. 

Gly-Pro-Ala was used as a substrate for dipeptidyl peptidase IV and was separated from Gly-Pro on a Zorbax ODS (15 cm x 4.6 mm) column by means of a mobile phase containing 10 mAf KH2P04(pH 2.5) and 1.0 mM 1-octanesulfonate as an ion-pairing agent. The substrate used for amino peptidase-P was Gly-Pro-Hyp, which was separated from Pro-Hyp by means of a mobile phase containing a 15 85 (v/v) mixture of (10 mM KH2P04) (pH 2.5) and 1-octanesulfonate) and acetonitrile. The eluate was monitored at 210 nm. 

The reaction mixture contained in a final volume of 500 fiL 0.03 mmol Tris-HCl (pH 8.0), 0.3 /imol of peptide substrate, and the appropriate amount of microvillous fraction, which was prepared from rabbit kidney. In the aminopeptidase-P assay, 1.0 /imol of manganese chloride was also included. Reactions were run for 30 minutes at 37°C and were stopped by adding 400 fiL of 10% perchloric acid. After centrifugation, 20 / L aliquots were injected. The amount of product formed was linear with amount of enzyme added out to 250 jug and 1000 ng of microvillus protein for dipeptidyl peptidase IV and amino peptidase-P, respectively. 

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MMP-11 Stromelysin 3 Secreted MMP-11 shows more similarity to the MT-MMPs, is convertase-activatable and is secreted therefore usually associated to convertase-activatable MMPs. 

Examples of the application of SAR-by-NMR include the design of stromelysin and human papillomavirus E2 protein inhibitors [7, 8]. 

Olejniczak ET, Hajduk PJ, Marcotte PA et al (1997) Stromelysin inhibitors designed from weakly bound fragments effects of linking and cooperativity. J Am Chem Soc 119 5828-5832... 

Winyard, P.G., Zhang, Z., Chidwick, K., Blake, D.R., Carrell, R.W. and Murphy, G. (1991). Proteolytic inactivation of human ai-antitrypsin by human stromelysin. FEES Lett. 279, 91-93. 

Three-Dimensional Structure of the Inhibited Catalytic Domain of Human Stromelysin-1 by Heteronuclear NMR Spectroscopy... 

Figure 2 Domain structure of the MMPs 92 kDa gelatinase-A (MMP-2), 72 kDa gelatinase-B (MMP-9), the collagenases (MMP-1, -8, and -13), stromelysin-1 (MMP-3) and matrilysin (MMP-7). Matrilysin is the only known MMP that does not have a C-terminal hemopexin-like domain.

III. ASSIGNMENT OF THE RESONANCES OF THE INHIBITED CATALYTIC DOMAIN OF STROMELYSIN-1... 

For the complex of the inhibited catalytic domain of stromelysin-1, 2-D doubly filtered COSY and TOCSY experiments performed... 

An advantage of NMR spectroscopy is the analysis of protein dynamics. Measurement and analysis of the relaxation parameters R1 R2, and the 15N NOE of 15N-labeled proteins leads to an order parameter (S2) that can describe the relative mobility of the backbone of the protein. Both collagenase-1 and stromelysin-1 have been studied either as inhibited complexes or the free protein [19, 52], Stromleysin-1 was studied with inhibitors binding to prime or nonprime subsites. Presence or absence of inhibitors in the nonprime sites had minor effects on the highly ordered structure of residues in these subsites, which are in contact with the... 

Figure 12 Catalytic mechanism of thermolysin and stromelysin-1. (A) The mechanism of thermolysin [54], (B) The mechanism of stromleysin-1 [10]. Equivalent residues to Tyr-157 and His-231 are not observed for stromelysin-1. The proposed mechanism for collagenase-1 [S3] is similar to stromelysin-1, but also involves Asn-180 (equivalent to Asn-162 in stromelysin-1). This residue cannot participate in stromelysin-1 due to an additional residue between Ala-165 and Asn-162. (Adapted from Ref. 10.)...

Gooley PR, Johnson BA, Marcy AI, Cuca GC, Salowe SP, Hagmann WK, Esser CK, Springer JP. Secondary structure and zinc ligation of human recombinant short-form stromelysin by multidimensional heteronuclear NMR. Biochemistry 1993 32 13098-13108. 

Becker JW, Marcy AI, Rokosz LL, Axel MG, Burbaum JJ, Fitzgerald PMD, Cameron PM, Esser CK, Hagmann WK, Hermes JD, Springer JP. Stromelysin-1 Three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme. Prot Sci 1995 4 1966-1976. 

Marcy AI, Eiberger LL, Harrison R, Chan HK, Hutchinson NI, Hagmann WK, Cameron PM, Boulton DA, Hermes JD. Human fibroblast stromelysin catalytic domain expression, purification and characterization of a C-terminally truncated form. Biochemistry 1991 30 6476-6483. 

Van Doren SR, Kurochkin AV, Hu W, Ye Q-Z, Johnson LL, Hupe DJ, Zuiderweg ERP. Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor. Prot Sci 1995 4 2487-2498. 

Stockman BJ, Waldon DJ, Gates JA, Schaill TA, Kloosterman DA, Mizsak SA, Jacobsen EJ, Belonga KL, Mitchell MA, Mao B, Petke JD, Goodman L, Powers EA, Ledbetter SR, Kaytes PS, Yogeli G, Marshall VP, Petzold GL, Poorman RA. Solution structure of stromelysin complexed to thiadiazole inhibitors. Prot Sci 1998 7 2281-2286. 

Yuan P, Marshall VP, Petzold GL, Poorman RA, Stockman BJ. Dynamics of stromelysin/inhibitor interactions studied by 15N NMR relaxation measurements Comparison of ligand binding to the SrS3 and S -Sy subsites. J Biomol NMR 1999 15 55-64. 

Hajduk PJ, Sheppard G, Nettesheim DG, Olejniczak ET, Shuker SB, Meadows RP, Steinman DH, Carrera GM, Marcotte PA, Severin J, Walter K, Smith H, Gubbins E, Simmer R, Holzman TF, Morgan DW, Davidsen SK, Summers JB, Fesik SW. Discovery of potent nonpeptide inhibitors of stromelysin using SAR by NMR. J Am Chem Soc 1997 119 5818-5827. 

A common way to benefit from the ability to combine different molecular orbital methods in ONIOM is to combine a DFT or ab-initio description of the reactive region with a semi-empirical treatment of the immediate protein environment, including up to 1000 atoms. Due to the requirement for reliable semi-empirical parameters, as discussed in Section 2.2.1, this approach has primarily been used for non-metal or Zn-enzymes. Examples include human stromelysin-1 [83], carboxypeptidase [84], ribonucleotide reductase (substrate reaction) [85], farnesyl transferase [86] and cytosine deaminase [87], Combining two ab-initio methods of different accuracy is not common in biocatalysis applications, and one example from is an ONIOM (MP2 HF) study of catechol O-methyltransferase [88],... 

The observed normal isotope effect of 1.9 provides further evidence supporting the role of Asp55 as the general base. Namely, a normal isotope effect of 1.9 is most consistent with general base catalysis by an amino acid side chain, as inverse isotope effects are commonly observed when a zinc-bound water molecule, or hydroxide, is the attacking nucleophile. For example, the zinc-containing enzymes AMP deaminase [111], thermolysin [112], stromelysin [113], and a desuccinylase [114] are each believed to utilize a zinc-bound water as the nucleophile, and all of these reactions are characterized by an inverse deuterium isotope effect. This inverse isotope effect is thought to result from a dominant... 

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