2´-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon intermediate of the ribulose bisphosphate carboxylase reaction

Abstract

The six-carbon intermediate of the ribulose 1,5-bisphosphate (RuBP) carboxylase reaction, 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (CKABP), was prepared enzymatically by quenching the reaction with acid after a short time ( ca 12 ms). Over a wide pH range (4-11), GKABP undergoes a slow ( t 1/2 = 1 h), pH-independent decarboxylation. No detectable decomposition of CKABP occurs over a six-week period at — 80 °C. The decarboxylation of CKABP is acid-catalysed and is also catalysed by deactivated enzyme lacking the activator carbamate-divalent metal ion complex. Decarboxylation is accompanied by β-elimination of the C-1 phosphate from the 2,3-enediolate. Under alkaline conditions (pH >11) CKABP undergoes hydrolysis. Non-enzymatic hydrolysis of the intermediate is also accompanied by β-elimination of the C-1 phosphate (presumably from the aci-acid of the upper glycerate 3-phosphate) and the formation of pyruvate. Fully activated enzyme catalyses the complete hydrolysis of CKABP to glycerate 3-phosphate, although enzymic hydrolysis of CKABP is limited by an event not on the direct path of carboxylation. Carbon-13 NMR analysis of [2',3- 13 C]CKABP indicates that it exists in solution predominantly (> 85%) as the C-3 ketone. In contrast, borohydride trapping of CKABP formed from [3- 18 O]RuBP indicates that the intermediate exists on the enzyme predominantly (> 94%) as the hydrated C-3 gem-diol. In solution, the hydration of the C-3 ketone of CKABP proceeds slowly ( k = 2.5 x 10 -3 s -1 ). The enzymatic hydration of CKABP must proceed at least as fast as k cat ( ca. 5 s -1 ) or at least 2000 times faster than the hydration of CKABP in solution.