Alkylation of glyceraldehyde-3-phosphate dehydrogenase with haloacetylphosphonates. An unusual pH-dependence.
Two new alkylating reagents, chloro- and bromo-acetylphosphonate, were found to be very effective thiol-blocking reagents. The pH-dependence of the reaction of BAP with 2,4-dinitrothiophenol (25 degrees C, I 0.5) shows a tailing bell-shaped curve (with a plateau at high pH) characteristic of two ionizing groups: the thiol group (pKa 3.2) and the phosphonate group (pKa2 4.6). The rate constant for the reaction of the monoanionic inhibitor with dinitrothiophenolate (k2 = 7 M-1.s-1) is 120 times larger than that of the dianionic species. The haloacetylphosphonates were found to be irreversible inhibitors of glyceraldehyde-3-phosphate dehydrogenase from a variety of sources. They react with the active-site thiol group (Cys-149) and are half-site reagents with yeast glyceraldehyde-3-phosphate dehydrogenase. Thus, when two of the identical four subunits are modified the enzyme is catalytically inactive. The effects of pH (7-10), 2H2O and NAD+ on the reaction with the yeast enzyme were examined in detail. NAD+ enhances the alkylation rates. The second-order rate constant does not show a simple sigmoidal dependence on pH but rather a tailing bell-shaped curve (pKa 7.0 and 8.4) qualitatively similar to that obtained with dinitrothiophenol. There is no significant solvent isotope effect on the limiting rate constants and a normal isotope effect on the two pKa values. The results are consistent with the more reactive enzyme species containing a thiolate and an acidic group that may either donate a proton to the dianionic haloacetylphosphonate or orient the inhibitor.
Li
Y K
YK
Department of Chemistry, Tulane University, New Orleans, LA 70118.
Boggaram
J
J
Byers
L D
LD
D019297:2,4-Dinitrophenol; D000478:Alkylation; D004140:Dinitrophenols; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013816:Thermodynamics
D004140:Dinitrophenols; D007202:Indicators and Reagents; D009943:Organophosphorus Compounds; D009243:NAD; C069016:chloroacetylphosphonate; C069017:bromoacetylphosphonate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019297:2,4-Dinitrophenol