Rubisco: Characteristics of misfire during catalysis and the properties of the substrate binding sites.

Abstract

The binding of carboxy-arabinitol bisphosphate (CABP), carboxy-arabinitol 1-phosphate (CA1P), carboxy-ribitol bisphosphate (CRBP), to carbamylated sites or xylulose bisphosphate (XuBP) and ribulose bisphosphate (RuBP) to decarbamylated sites on ribulose bisphosphate carboxylase/oxygenase (rubisco) exhibits negative cooperativity. The binding of ligands to decarbamylated sites was highly pH-dependent between 7.5 and 8.5. Lower pH enhanced binding affinity. The binding of ligands to carbamylated sites was pH-independent. A binding model for negative cooperative interactions among catalytic sites is proposed based on the observations and the crystallographic structure of rubisco. Fully activated, purified rubisco slowly loses its activity during in vitro catalysis after exposure to RuBP. This time-dependent kinetics is termed as "fallover". Two different fallover patterns were demonstrated during CO₂ fixation, one with little loss of activator CO₂ at pH 8.5 and the second with loss of activator CO₂ at pH 7.5. The two inhibitors being produced during fallover were isolated by high performance anion exchange chromatography (HPAE) and identified as XuBP and 3-ketoarabinitol 1,5-bisphosphate by pulsed amperometric detection (PAD) either directly or after reduction by NaBH₄. Because of the weak binding affinity of XuBP to catalytic sites of rubisco at pH 8.5, there was little loss of activator CO₂ during fallover. 3-keto-arabinitol-P₂ which binds to carbamylated rubisco sites became the major inhibitor at that pH. However, at pH 7.5, the binding of XuBP to decarbamylated sites can cause a shift in equilibrium with loss of carbamylated sites even in the presence of excess CO₂ and Mg²⁺, resulting in the loss of activator CO₂ during fallover. XuBP was isolated and identified from celery leaves by HPAE-PAD. A new method was developed for the determination of the substrate specificity of rubisco. It is based on the specific ¹⁴C-labeling of 3-phosphoglycerate (PGA) from the carboxylase reaction and its dilution from the oxygenase reaction. Therefore, the ratio of carboxylation to oxygenation can be measured directly by determining the specific radioactivity of PGA produced from both reactions with HPAE-PAD separation of the total PGA and scintillation counting of ¹⁴C-labeled PGA.