Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom.
Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an alpha-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an alpha-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.
Pretzer
D
D
Merck, Sharp, and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Schulteis
B
B
Vander Velde
D G
DG
Smith
C D
CD
Mitchell
J W
JW
Manning
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D000933:Antifibrinolytic Agents / Q000494:pharmacology; D001665:Binding Sites; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000201:enzymology*; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000187:drug effects; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D000933:Antifibrinolytic Agents; D003435:Crotalid Venoms; D005343:Fibrinolytic Agents; D004492:Edetic Acid; D008666:Metalloendopeptidases; C058789:fibrolase; D015032:Zinc