Structural Characterization of the Catalytic Active Site in the Latent and Active Natural Gelatinase B from Human Neutrophils

Abstract

Matrix metalloproteinases are endopeptidases that have a leading role in the catabolism of the macromolecular components of the extracellular matrix in a variety of normal and pathological processes. Human gelatinase B is a zinc-dependent proteinase and a member of the matrix metalloproteinase family that is involved in inflammation, tissue remodeling, and cancer. We have conducted x-ray absorption spectroscopy, atomic emission, and quantum mechanics studies of natural and activated human gelatinase B. Our results show that the natural enzyme contains one catalytic zinc ion that is central to catalysis. In addition, upon enzyme activation, the catalytic zinc site exhibits a conformation change that results in the expansion of the bond distances around the zinc ion and the replacement of one sulfur with oxygen. Interestingly, quantum mechanics calculations show that oxygen ligation at the catalytic zinc ion exhibits a greater affinity to the binding of an oxygen from an amino acid residue rather than from an external water molecule. These results suggest that the catalytic zinc ion plays a key role in both substrate binding and catalysis.Remodeling of the extracellular matrix is an important event in many normal and pathological processes such as growth, wound repair, tumor metastasis, and leukocyte mobilization in inflammation. A large family of zinc-dependent proteinases, the matrix metalloproteinases (MMPs), 1 is considered to be primarily responsible for this matrix catabolism (1-4). Details of their variety and substrate specificity are documented in several recent reviews (5-7). The MMPs consist of a large family of proteinases that share many common structural and functional elements. MMPs can be categorized based on amino acid sequence similarity, substrate specificity, and domain structure. They include collagenases, which cleave triple helical interstitial collagens; gelatinases, which cleave denatured collagen, elastin and type IV and V collagens; stromelysins, which mainly cleave proteoglycans; and membrane-type MMPs, which are associated with the activation of pro-MMPs (2, 3). In addition to these classical MMPs, other metalloproteinases, mainly with functions other than matrix catabolism, are being discovered at an increasing pace. All MMPs are synthesized as preproenzymes and are usually secreted as inactive pro-MMPs. The primary structure of each MMP is composed of some of the following domain motifs: a signal peptide, a propeptide, a catalytic domain that contains a zinc ion central to catalysis, a linker, a hemopexin-like domain, a fibronectin type II domain, a transmembrane region and a cytoplasmic domain (found in membrane-types 1-6), a furinrecognition sequence, and a vitronectin-like domain (7). Moreover, each member of the MMP family is characterized by a different domain composition, but contains a propeptide domain of about 80 amino acids with a conserved PRCGVPDV motive that ligates to the catalytic zinc ion via the cysteine residue to maintain the latency of the proenzyme (8, 9). The ca...

Reduced nonprotein thiols inhibit activation and function of MMP-9: Implications for chemoprevention

“…5 demonstrates the feasibility of NAC docking at MMP-9's active-site Zn molecule. This premise is supported by the studies of Kleifeld et al, who clarified the nature of MMP-9 activation, i.e., the latent Zn binding site becomes catalytically competent by dissociation of the thiol propeptide from the Zn atom [30]. Although our data suggest a plausible MMP-9 inhibitory mechanism, in order to provide conclusive evidence for GSH's and NAC's interactions with MMP-9's active site, it is necessary to conduct quantitative biophysics.…”

Simultaneous measurement of MMP9 and TIMP1 mRNA in human non small cell lung cancers by multiplex real time RT-PCR

“…Because of the ability to degrade the major component of the ECM and basement membrane, its overexpression facilitates the metastatic spread [11]. Growth factors (TGF␣, TGF␤, TNF␣, EGF), cytokines (interleukin 1 and 4), and hormones (progesterone, retinoids) are responsible for the pre-translational control of MMP9 expression [11,12]. At the post-translational level, all MMPs are under the control of specific tissue inhibitors of metalloproteinases (TIMPs) that bind in proximity of the catalytic domain of MMPs, preventing substrate attachment [13].…”

X-ray Absorption Studies of Human Matrix Metalloproteinase-2 (MMP-2) Bound to a Highly Selective Mechanism-based Inhibitor

“…The zinc-protein ratio was further investigated by XAS edge step analysis following previous procedures (32). Briefly, the edge step of the XAS coefficient should be proportional to the concentration of the absorbing element.…”

“…4, although we carried out energy minimizations with both stereoisomers. (32) suggest that full-length intact MMPs (for the ones tested) contain a single zinc ion, while truncated enzymes (lacking the C-terminal domain) contain two zinc ions. In addition, it was suggested that the zinc content in MMPs might be dependent on the procedure used for enzyme purification (32,33).…”

“…(32) suggest that full-length intact MMPs (for the ones tested) contain a single zinc ion, while truncated enzymes (lacking the C-terminal domain) contain two zinc ions. In addition, it was suggested that the zinc content in MMPs might be dependent on the procedure used for enzyme purification (32,33). Nevertheless, the crystal structure of an inactive fulllength pro-MMP-2 mutant clearly shows the presence of both structural and catalytic zinc ion sites (13).…”