o-Phthalaldehyde, a fluorescence probe of aldolase active site.
Conditions were determined in which approximately one mole of omicron-phthalaldehyde reacts with one mole of aldolase subunit yielding a stable fluorescent isoindole derivative. During this chemical modification, a linear relationship was observed between the enzyme inactivation and absorbance change (337 nm) or fluorescence change (lambda em 420 nm, and lambda ex 338 nm) characteristic for isoindole ring formation. The reaction follows second-order kinetics, k = 1.1 X 10(3) M-1 S-1, in 50 mM borate buffer, pH 8.4 at 25 degrees C. The modification of aldolase results in loss of approximately one -SH group per protein subunit. The enzyme is protected against modification by substrates and competitive inhibitors. Essentially no isoindole derivative is formed when the glycerol-1-phosphate-lysyl derivative of aldolase is used for modification studies. It is concluded that aldolase modification occurs at the active-site region. Isolation of cross-linked peptides suggests that Lys-227 and Cys-336 are involved in formation of the isoindole derivative. This result supports Cys-336 as the active-site cysteine necessary for aldolase catalytic activity. Fluorescence studies have shown that the isoindole group linked to aldolase has its lambda max, em markedly shifted toward shorter wavelength in comparison to the fluorescence of free isoindole derivatives in aqueous solution. In model studies a linear relationship between lambda max, em of 1-(beta-hydroxyethylthio)-2-beta-hydroxyethylisoindole and the solvent polarity or acidity was observed. The results of the studies suggest that the microenvironment of the cleft in aldolase which binds isoindole appears to be of low acidity and low polarity. The apparent low polarity experienced by the isoindole probe may be due to its location in an actual low-polarity portion of the active site, or may be due to non-relaxing surroundings of the probe.
D000447:Aldehydes; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D005456:Fluorescent Dyes; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D009764:o-Phthalaldehyde
D000447:Aldehydes; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; D009764:o-Phthalaldehyde; D005634:Fructose-Bisphosphate Aldolase