Metal ion binding and activation of Streptomyces griseus dinuclear aminopeptidase: cadmium(II) binding as a model.
A detailed metal binding and activation of the dinuclear aminopeptidase from Streptomyces griseus (sAP) has been analyzed and modeled by means of metal titration as well as kinetic and thermodynamic techniques using Cd2+ as a probe. Cd2+ binds to the two metal-binding sites in a sequential manner to produce a very active Cd2+-substituted derivative, particularly in the presence of Ca2+ (53% and 90%, respectively, relative to the activities of the native form in terms of kcat/Km under the same conditions). The first stepwise formation constant for the binding of metal to the dinuclear site (to form M-sAP) was found to determine the metal-binding selectivity, regardless of the magnitude of the second stepwise formation constant (to form M,M-sAP from M-sAP). Interestingly, despite the seemingly very different binding profiles for different metal ions under different conditions, all of them can be well described and fitted by the sequential binding model. In addition, Ca2+ was found to significantly affect metal binding, inhibition, and entropy of activation of this enzyme, and its role in sAP action is re-evaluated.
Hasselgren
C
C
Department of Chemistry and Institute for Biomolecular Science, University of South Florida, Tampa 33620-5250, USA.
Park
H I
HI
Ming
L J
LJ
D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008024:Ligands; D019216:Metals, Heavy / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013305:Streptomyces griseus / Q000201:enzymology*; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D001426:Bacterial Proteins; D008024:Ligands; D019216:Metals, Heavy; D002104:Cadmium; D003035:Cobalt; D000626:Aminopeptidases; D015032:Zinc; D002118:Calcium