Myo-inositol monophosphatase: binding of terbium and a cross-linking reagent to the catalytic site cavity.
In the presence of myo-inositol monophosphatase, terbium ions can be excited by energy transfer from the aromatic side chains of the protein. This enhancement of Tb3+ luminescence due to its binding to the enzyme at pH 6.5 was used to determine the dissociation constant (56 microM) for the monophosphatase-Tb3+ complex. Competition luminescence studies also indicated that calcium ions could compete with terbium ions for binding to the enzyme with a dissociation constant of 200 microM. Conditions were determined in which approximately 1 mol of o-phthaldehyde reacts with one subunit of the dimeric protein, yielding a stable fluorescent isoindole derivative. The kinetics of the reaction, monitored by fluorescence spectroscopy, yields a second-order rate constant (K2 = 110 M-1 s-1). The inactivation of the monophosphatase by o-phthaldehyde has been shown to be protected by the substrate beta-glycerolphosphate. Essentially, no formation of any isoindole derivative is detected after one sulfhydryl residue of the enzyme is reacted with N-ethylmaleimide. It is suggested that the site of the isoindole chromophore formed upon reaction of the enzyme with o-phthaldehyde includes one lysyl and one cysteinyl residue located in the cavity of the catalytic site.
Kwok
F
F
Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic.
Wang
X
X
Churchich
J E
JE
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D008961:Models, Structural; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013725:Terbium / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D009764:o-Phthalaldehyde / Q000494:pharmacology
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D013725:Terbium; D009764:o-Phthalaldehyde; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D002118:Calcium