Identification of the (183)RWTNNFREY(191) region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity.

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.

Tissue inhibitors of metalloproteinases: evolution, structure and function.

The matrix metalloproteinases (MMPs) play a key role in the normal physiology of connective tissue during development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. An important mechanism for the regulation of the activity of MMPs is via binding to a family of homologous proteins referred to as the tissue inhibitors of metalloproteinases (TIMP-1 to TIMP-4). The two-domain TIMPs are of relatively small size, yet have been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs, proMMP activation, cell growth promotion, matrix binding, inhibition of angiogenesis and the induction of apoptosis. Mutations in TIMP-3 are the cause of Sorsby's fundus dystrophy in humans, a disease that results in early onset macular degeneration. This review highlights the evolution of TIMPs, the recently elucidated high-resolution structures of TIMPs and their complexes with metalloproteinases, and the results of mutational and other studies of structure-function relationships that have enhanced our understanding of the mechanism and specificity of the inhibition of MMPs by TIMPs. Several intriguing questions, such as the basis of the multiple biological functions of TIMPs, the kinetics of TIMP-MMP interactions and the differences in binding in some TIMP-metalloproteinase pairs are discussed which, though not fully resolved, serve to illustrate the kind of issues that are important for a full understanding of the interactions between families of molecules.

Molecular structure of 5-methyl thiophene acryloyl ethyl thiolester: a vibrational spectroscopic and density functional theory study.

Enzyme-substrate intermediates involving the acyl group 5-methyl thiophene acryloyl (5-MTA) bound to the active site of an enzyme via a sulfur or selenium atom have been characterized by Raman spectroscopy (e.g., J. D. Doran and P. R. Carey, Biochemistry 1996, 35, 12495-12502, and M. J. O'Connor et al., J Amer Chem Soc 1996, 118, 239-240). Raman difference spectra reveal the Raman spectrum of the acyl group in the active site and, in turn, these can be used to probe acyl group conformation and active site forces and interactions. In order to improve the understanding of the relationship between conformational states and vibrational spectra of 5-MTA thiolesters, calculations based on a density functional theory analysis are undertaken for 5-methyl thiophene acryloyl ethyl ester. The calculations provide the precise geometries and energies of rotomers of 5-MTA ethyl thiolester involving rotational isomerism about the C--C single bonds flanking the ethylenic linkage and the S--C bond linking the ethyl group to the sulfur atom. The calculations also provide the vibrational spectrum for each conformer and these predictions are compared with the experimental Raman an IR data for the thiolester in carbon tetrachloride. Modes are identified that can act as conformational markers for isomerism about the C--C and S--C2H5 single bonds. These findings are used to identify the two conformational states giving rise to the Raman spectrum of the 5-MTA-S-enzyme formed by the viral cysteine protease HAV-3C.

Electric fields in active sites: substrate switching from null to strong fields in thiol- and selenol-subtilisins.

Although known to be important factors in promoting catalysis, electric field effects in enzyme active sites are difficult to characterize from an experimental standpoint. Among optical probes of electric fields, Raman spectroscopy has the advantage of being able to distinguish electronic ground-state and excited-state effects. Earlier Raman studies on acyl derivatives of cysteine proteases [Doran, J. D., and Carey, P. R. (1996) Biochemistry 35, 12495-502], where the acyl group has extensive pi-electron conjugation, showed that electric field effects in the active site manifest themselves by polarizing the pi-electrons of the acyl group. Polarization gives rise to large shifts in certain Raman bands, e.g. , the C=C stretching band of the alpha,beta-unsaturated acyl group, and a large red shift in the absorption maximum. It was postulated that a major source of polarization is the alpha-helix dipole that originates from the alpha-helix terminating at the active-site cysteine of the cysteine protease family. In contrast, using the acyl group 5-methylthiophene acryloyl (5-MTA) as an active-site Raman probe, acyl enzymes of thiol- or selenol-subtilisin exhibit no polarization even though the acylating amino acid is at the terminus of an alpha-helix. Quantum mechanical calculations on 5-MTA ethyl thiol and selenol ethyl esters allowed us to identify the conformational states of these molecules along with their corresponding vibrational signatures. The Raman spectra of 5-MTA thiol and selenol subtilisins both showed that the acyl group binds in a single conformation in the active site that is s-trans about the =C-C=O single bond. Moreover, the positions of the C=C stretching bands show that the acyl group is not experiencing polarization. However, the release of steric constraints in the active site by mutagenesis, by creating the N155G form of selenol-subtilisin and the P225A form of thiol-subtilisin, results in the appearance of a second conformer in the active sites that is s-cis about the =C-C=O bond. The Raman signature of this second conformer indicates that it is strongly polarized with a permanent dipole being set up through the acyl group's pi-electron chain. Molecular modeling for 5-MTA in the active sites of selenol-subtilisin and N155G selenol-subtilisin confirms the findings from Raman spectroscopic studies and identifies the active-site features that give rise to polarization. The determinants of polarization appear to be strong electron pull at the acyl carbonyl group by a combination of hydrogen bonds and the field at the N-terminus of the alpha-helix and electron push from a negatively charged group placed at the opposite end of the chromophore.

Active site properties of the 3C proteinase from hepatitis A virus (a hybrid cysteine/serine protease) probed by Raman spectroscopy.

Although the HAV 3C proteinase is a cysteine protease, it displays an active site configuration which resembles mammalian serine proteases and is structurally distinct from the papain superfamily of thiol proteases. Given the interesting serine/cysteine protease hybrid nature of HAV 3C, we have probed its active site properties via the Raman spectra of the acyl enzyme, 5-methylthiophene acryloyl HAV 3C, using the C24S variant of the enzyme to obtain stoichiometric acylation. The Raman difference spectral data show that the major population of the acyl groups in the active site experiences electron polarization intermediate between that in the papain superfamily and that in a nonpolarizing site. This is evidenced by the values of the acyl group ethylenic stretching frequency which occur near 1602 cm(-1) in a nonpolarizing environment, at 1588 cm(-1) when bound to HAV 3C (C24S), and at 1579 cm(-1) in acyl papains. The value of the electronic absorption maximum for the HAV 3C (C24S) acyl enzyme and the deacylation rate constant fit the correlation developed for the papain superfamily, suggesting that for HAV 3C too, polarizing forces in the active site can contribute to rate acceleration via transition state stabilization. The major population in the active site is s-cis about the acyl group's C1-C2 bond, but there is a second population that is s-trans, and this secondary population is not polarized. The two populations are evidenced by the presence of two sets of marker bands for s-cis and s-trans in the Raman spectra, which occur principally in the C=C stretching region near 1600 cm(-1), in the C-C stretching region near 1100 cm(-1), and near 560 cm(-1). The positions of the acyl carbonyl features in the Raman spectra point to hydrogen-bonding strengths of 20-25 kJ mol(-1) between the C=O and H-bonding donors in the active site. The 5-methylthiophene acryloyl HAV 3C (C24S) is a relatively unreactive acyl enzyme, deacylating with a pKa of 7.1 and a rate constant of 0.000 31 s(-1) at pH 9. Unlike most other cysteine or serine protease acyl enzymes characterized by Raman spectroscopy, no changes in the Raman spectrum could be detected with changes in pH.