Xylulose 1,5-bisphosphate synthesized by ribulose 1,5-bisphosphate carboxylase/oxygenase during catalysis binds to decarbamylated enzyme

Abstract

Xylulose 1,5-bisphosphate (XuBP) is synthesized from ribulose 1,5-bisphosphate (RuBP) at carbamylated catalytic sites on ribulose 1,5-bisphosphate carboxylase (Rubisco) with significant amounts of XuBP being formed at pH less than 8.0. XuBP has been separated by high performance liquid chromatography and identified by pulsed amperometry from compounds bound to Rubisco during catalysis with the purified enzyme and from celery (Apium graveolens var Utah) leaf extracts. XuBP does not bind tightly to carbamylated sites, but does bind tightly to decarbamylated sites. Upon incubation of fully activated Rubisco with 5 micromolar XuBP, loss of activator CO2 occurred before XuBP bound to the enzyme catalytic sites, even in the presence of excess CO2 and Mg2+. Binding of XuBP to decarbamylated Rubisco sites was highly pH dependent. At pH 7.0 and 7.5 with 10 millimolar MgCl2 and 10 millimolar KHCO3, the apparent dissociation constant for XuBP, Kd, was 0.03 micromolar, whereas at pH 8.0 and 8.5, the apparent Kd was 0.35 and 2.0 micromolar, respectively. This increase in Kd with pH was a result of a decrease in the association rate constant and an increase in the dissociation rate constant of XuBP bound to decarbamylated sites on Rubisco. The Kd of 2-carboxyarabinitol 1-phosphate binding to carbamylated sites was only slightly pH dependent.