Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase.
Trinitrobenzene sulfonate rapidly inactivates ribulosebisphosphate carboxylase/oxygenase from both spinach and Rhodospirillum rubrum. With large molar excesses of the reagent, the reactions obey pseudo-first order kinetics and the rates of inactivations are directly proportional to the concentrations of trinitrobenzene sulfonate; thus, there is no indication of reversible complexation of reagent with enzyme. Saturating levels of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduce the rates of inactivations but do not prevent them, thereby suggesting that the groups subject to arylation remain accessible in the enzyme complexed with competitive inhibitor. Characterization of tryptic digests of the inactivated enzymes reveals that Lys-166 of the R. rubrum enzyme and Lys-334 of the spinach enzyme are the only major sites of arylation. Both of these lysines have been assigned to the catalytic site by prior affinity labeling studies and are found within highly conserved regions of primary structure. As a monoanion over a wide pH range, trinitrobenzene sulfonate, for which the carboxylase lacks high affinity, can thus be used to determine the pKa values of the two active-site lysyl epsilon-amino groups. Based on the pH dependency of inactivation of the R. rubrum enzyme by trinitrobenzene sulfonate, the epsilon-amino group of Lys-166 exhibits a pKa of 7.9 and an intrinsic reactivity (ko) of 670 M-1 min-1. In analogous experiments, Lys-334 of the spinach enzyme exhibits a pKa of 9.0 and a ko of 4500 M-1 min-1. Under deactivation conditions (i.e. in the absence of CO2 and Mg2+), the pKa of Lys-334 becomes 9.8 and the ko is increased to 26,000 M-1 min-1. By comparison, the reaction of trinitrobenzene sulfonate with N-alpha-acetyl-lysine reveals a pKa of 10.8 and a ko of 1250 M-1 min-1. The spinach carboxylase, catalytically inactive as a consequence of selective arylation of Lys-334, still exhibits tight binding of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate. Therefore, Lys-334 is not required for substrate binding and may serve a role in catalysis. The unusually low pKa of Lys-166 argues that this residue is also important to catalysis rather than substrate binding.
D000838:Anions; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010428:Pentosephosphates; D010446:Peptide Fragments / Q000032:analysis; D010944:Plants / Q000201:enzymology; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013402:Sugar Alcohols; D013403:Sugar Phosphates / Q000378:metabolism; D014301:Trinitrobenzenes / Q000378:metabolism; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology; D014357:Trypsin
D000838:Anions; D010428:Pentosephosphates; D010446:Peptide Fragments; D013402:Sugar Alcohols; D013403:Sugar Phosphates; D014301:Trinitrobenzenes; C002315:N(alpha)-acetyllysine; C032687:2-carboxyarabinitol 1,5-biphosphate; D014302:Trinitrobenzenesulfonic Acid; D010105:Oxygenases; D014357:Trypsin; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine