Mechanism of Ca2+-dependent activity of human neutrophil gelatinase B.

Progelatinase B can be activated in vitro by organomercurial compounds and by proteolytic enzymes such as trypsin, chymotrypsin, and stromelysin. Activation of the proenzyme by either 4-aminophenylmercuric acetate or chymotrypsin yielded proteins that absolutely required Ca2+ for activity, regardless of the pH of the reaction mixture. The trypsin- and stromelysin-activated gelatinases, on the other hand, did not require Ca2+ for activity at pH 7.5, but the activity of the trypsin-activated enzyme became Ca2+ dependent as the pH increased. The pH study revealed that an amino acid residue with an apparent pKa of 8.8 was involved in this process. The NH2-terminal analyses showed that trypsin- and stromelysin-activated enzymes had the same NH2 termini (Phe88), but 4-aminophenylmercuric acetate- and chymotrypsin-activated enzymes had Met75 and Gln89 or Glu92 as the NH2-terminal amino acid, respectively. These data, in conjunction with the x-ray crystal structure of collagenase, suggest that a salt linkage involving Phe88 is responsible for the Ca2+-independent activity of trypsin- and stromelysin-activated gelatinase. Replacing Asp432 in progelatinase with either Glu, Asn, Gly, or Lys resulted in the proteins that, upon activation by trypsin, required Ca2+ for activity. These substitutions did not significantly affect Km for the synthetic substrate but decreased the kcat and increased the half-maximal Ca2+ concentration required for enzyme activity (KCa) by severalfold. The effects on kcat and KCa depended on both charge and size of the side chains of the substituted amino acids. The decrease in kcat correlated well with the increase in KCa of the mutants. The orders of decrease in kcat and increase in KCa were wild type >/= D432E > D432N > D432G > D432K and wild type

Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases.

Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence. 400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate by 79%. The wild-type and D432G and D433E, mutant enzymes had similar Km values for the synthetic substrate and similar Ki values for the competitive inhibitor, GM6001. The kcat/Km values for D432G and D433E mutant enzymes, however, were reduced by a factor of approximately 4 and their KaCa values were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.

Mechanism of activation of human neutrophil gelatinase B. Discriminating between the role of Ca2+ in activation and catalysis.

Gelatinase B is a Zn(2+)- and Ca(2+)-dependent endopeptidase that is secreted from cells as an inactive proenzyme. The enzyme can be activated in vitro by organomercurial compounds and by trypsin. The role of Ca2+ in autoproteolytic processing initiated by 4-aminophenylmercuric acetate and trypsin and in catalytic activity of the activated enzyme was investigated by zymography and by kinetic analysis. Treatment of unglycosylated 57.5-kDa pro-gelatinase B with 4-aminophenylmercuric acetate (1 mM) in the absence of Ca2+ generated a 49-kDa inactive intermediate (E'), whereas a 41.5-kDa active species (E") was generated in the presence of Ca2+ (5 mM). Upon addition of Ca2+ to the reaction mixture of Ca(2+)-depleted E' or E" at 37 degrees C, E' showed a lag period in generation of the product as a function of time, but E" presented an immediate activity. The appearance of enzymatic activity of E' correlated with the generation of the E" species. NH2-terminal sequence analyses showed that E' and E" had the same NH2 termini, i.e. Met-75, suggesting that Ca(2+)-dependent removal of COOH terminus of E' is required for activation of the enzyme. Treatment of pro-gelatinase B with trypsin in the absence of Ca2+, led to degradation of the enzyme. In the presence of Ca2+, trypsin processed the pro-enzyme to a 40-kDa active species. In contrast to E", this active species did not require Ca2+ for activity. The Ca2+ dependence of E" activity was also abolished by treatment of the enzyme with trypsin. NH2-terminal sequence analysis indicated that amino acid residues 75-87 had been removed from the NH2 terminus of E" by trypsin, suggesting that these residues are responsible for the Ca(2+)-dependent activity of the enzyme. Removal of Ca2+ and catalytic Zn2+ inhibited the activities of both E" and trypsin-treated E". In the absence of Ca2+, either Zn2+, Co2+, Mn2+, or Cd2+ was able to restore the activity of trypsin-treated E". None of the divalent cations tested however, was able to stimulate the activity of E" in the absence of Ca2+. These experiments further suggest that binding of Ca2+ to E" or removal of the NH2-terminal residues of the enzyme by trypsin induces a conformational change in the protein and makes the active site of the enzyme accessible to various metal ions rendering the enzyme active.

Stimulation of myosin light-chain kinase by Cd2+ and Pb2+.

The effect of Cd2+ on myosin light chain kinase (MLCK) reported in the literature is controversial, apparently because the level of Ca2+ contaminating the reaction mixture could not be accurately controlled by the addition of a metal chelator when Cd2+ was also present. In the present study, we have reduced the contaminating Ca2+ to a trace level that did not interfere with the enzyme activity; thus the use of a metal chelator was not necessary. We showed that Cd2+, or Pb2+ had a biphasic effect on MLCK isolated from chicken gizzard: stimulation at low and inhibition at high concentrations. (The stimulatory effect of on the enzyme activity isolated from chicken gizzard: stimulation at low and inhibition at high concentrations). The stimulatory effect of Cd2+ or Pb2+ on MLCK activity was not seen in the absence of calmodulin, and was abolished by trifluoperazine, a calmodulin antagonist, indicating that the heavy metals exert their activation via calmodulin. The inhibition of the enzyme activity by Cd2+ or Pb2+ at higher concentrations was also seen with the calmodulin-independent catalytic fragment of MLCK, suggesting that the inhibition is probably through their binding to sulfhydryl groups that are essential for catalytic activity. Pb2+ was more effective than Cd2+ in stimulating the enzyme activity, but less potent in inhibition. The extent of stimulation by heavy metals most likely resulted from a combination of the biphasic effects. Dithiothreitol and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine selectively chelated Cd2+ and Pb2+ over Ca2+, and reversed their stimulatory or inhibitory effect on MLCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of troponin C by Cd2+ and Pb2+.

Certain heavy metal actions such as Cd2+ and Pb2+ mimic Ca2+ effectively in stimulating calmodulin (CaM). We now show that these cations also activate skeletal muscle troponin C (TnC), a Ca2(+)-binding protein highly homologous to CaM. Like Ca2+, these cations allow TnC to alter its electrophoretic mobility on polyacrylamide gels, and to bind to phenyl-Sepharose. Moreover, they activate TnC to stimulate myofibrillar ATPase. When TnC was removed from the skeletal myofibrils by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), the ATPase activity was no longer stimulated by the cations. However, after reconstitution of CDTA-treated skeletal myofibril with TnC, the response of ATPase to Ca2+, Cd2+ or Pb2+ was restored. These findings suggest that the activation of myofibrillar ATPase by Cd2+ and Pb2+ is mediated through TnC. The ability of the heavy metals to stimulate TnC-supported ATPase activity correlated quite well with the ability to increase the extent of the myofibrillar superprecipitation. The activation of TnC by Cd2+ or Pb2+ could constitute a possible molecular basis for their toxicity.