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

Persistent Link:
http://hdl.handle.net/10150/185734
Title:
Rubisco: Characteristics of misfire during catalysis and the properties of the substrate binding sites.
Author:
Zhu, Genhai.
Issue Date:
1991
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
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.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Dissertations, Academic.; Botany.; Plant physiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Plant Sciences; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Jensen, Richard G.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleRubisco: Characteristics of misfire during catalysis and the properties of the substrate binding sites.en_US
dc.creatorZhu, Genhai.en_US
dc.contributor.authorZhu, Genhai.en_US
dc.date.issued1991en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe 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.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectDissertations, Academic.en_US
dc.subjectBotany.en_US
dc.subjectPlant physiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePlant Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorJensen, Richard G.en_US
dc.contributor.committeememberBourque, Don P.en_US
dc.contributor.committeememberBohnert, Hans J.en_US
dc.contributor.committeememberBartels, Paul G.en_US
dc.contributor.committeememberO'Leary, James W.en_US
dc.identifier.proquest9210338en_US
dc.identifier.oclc712066283en_US
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