ATP-dependent phosphoenolpyruvate carboxykinase (E. C. 4.1.1.49; PCK) is an enzyme that catalyses the reversible conversion of oxaloacetate and ATP into phosphoenolpyruvate, ADP and CO2. PCK is made up of about 500 to 600 amino acid residues and is divided into two roughly equal domains. Upon binding of substrates, the two domains of PCK move towards each other. PCK is well known for its role in gluconeogenesis but in some species, it can have an anaplerotic role. In other species, PCK is important for metabolic steps involved in fermentation.<p>Presented in this thesis are five solved crystal structures of the ATP-dependent form of PCK. Three of the PCK crystal structures determined were from <i>Escherichia coli</i>; one was a complex of ATP, Mg2+ and CO2, the second structure was an ATP, Mg2+, Mn2+, CO2 and oxaloacetate complex and, the third <i>E. coli</i> structure was a Lys213Ser mutant complexed with ATP, Mg2+and Mn2+. Two <i>Anaerobiospirillum succiniciproducens</i> PCK crystal structures were also solved; one was in the native form and the other was an ATP-Mg2+-Mn2+-oxalate complex. <p>In the <i>E. coli</i>-PCK-ATP-Mg2+-CO2 crystal complex structure, the observed location of CO2 was in agreement with a previously determined <i>E. coli</i> PCK-CO2 crystal structure, which incorporated CO2 into the structure by a different technique. The findings from the <i>E. coli</i> PCK-ATP-Mg2+-CO2 crystal structure allowed the reaction mechanism presented in this work to be proposed.<p>The PCK-ATP-Mg2+-Mn2+-CO2-oxaloacetate structure is the first structure where oxaloacetate is observed bound to PCK. Surprisingly, the observed location of oxaloacetate in this structure is 5 Angstroms away from its expected position near Mn2+. Oxaloacetate is weakly bound to a non-catalytic region of the enzyme. It is proposed that when the domains of PCK move towards each other upon binding nucleotide, oxaloacetate experiences steric crowding which results in it being pushed towards the active site to react. <p>Previous kinetic studies on the <i>E. coli</i> PCK mutant Lys213Ser have determined that Mn2+ is unexpectedly inhibitory. A crystal structure of K213S-PCK-ATP-Mg2+-Mn2+ demonstrates that Mn2+ is tetrahedrally coordinated in the active site, not octahedrally as occurred in other structures. By having Mn2+ in the tetrahedral coordination state, substrate binding in the active site of PCK is altered in a way that does not allow catalysis to occur.<p>The two crystal structures of <i>A. succiniciproducens</i> PCK were useful in quantifying the substrate-induced domain movement. A surface active site lid made up of residues 385 to 405 that had never been observed in any of the previous PCK crystal structures was observed in the <i>A. succiniciproducens</i> PCK-ATP-Mg2+-Mn2+-oxalate crystal structure. Mutational studies of this lid have shown it to be essential for the function of PCK; however, its exact function is not certain. It has been proposed that the lid has multiple functions. One is to sequester the substrates from bulk solvent. Another function may be to assist in domain closure. The third function may be to assist in the proper positioning of substrates in the active site.

ATP-dependent phosphoenolpyruvate carboxykinase (E. C. 4.1.1.49; PCK) is an enzyme that catalyses the reversible conversion of oxaloacetate and ATP into phosphoenolpyruvate, ADP and CO2. PCK is made up of about 500 to 600 amino acid residues and is divided into two roughly equal domains. Upon binding of substrates, the two domains of PCK move towards each other. PCK is well known for its role in gluconeogenesis but in some species, it can have an anaplerotic role. In other species, PCK is important for metabolic steps involved in fermentation.<p>Presented in this thesis are five solved crystal structures of the ATP-dependent form of PCK. Three of the PCK crystal structures determined were from <i>Escherichia coli</i>; one was a complex of ATP, Mg2+ and CO2, the second structure was an ATP, Mg2+, Mn2+, CO2 and oxaloacetate complex and, the third <i>E. coli</i> structure was a Lys213Ser mutant complexed with ATP, Mg2+and Mn2+. Two <i>Anaerobiospirillum succiniciproducens</i> PCK crystal structures were also solved; one was in the native form and the other was an ATP-Mg2+-Mn2+-oxalate complex. <p>In the <i>E. coli</i>-PCK-ATP-Mg2+-CO2 crystal complex structure, the observed location of CO2 was in agreement with a previously determined <i>E. coli</i> PCK-CO2 crystal structure, which incorporated CO2 into the structure by a different technique. The findings from the <i>E. coli</i> PCK-ATP-Mg2+-CO2 crystal structure allowed the reaction mechanism presented in this work to be proposed.<p>The PCK-ATP-Mg2+-Mn2+-CO2-oxaloacetate structure is the first structure where oxaloacetate is observed bound to PCK. Surprisingly, the observed location of oxaloacetate in this structure is 5 Angstroms away from its expected position near Mn2+. Oxaloacetate is weakly bound to a non-catalytic region of the enzyme. It is proposed that when the domains of PCK move towards each other upon binding nucleotide, oxaloacetate experiences steric crowding which results in it being pushed towards the active site to react. <p>Previous kinetic studies on the <i>E. coli</i> PCK mutant Lys213Ser have determined that Mn2+ is unexpectedly inhibitory. A crystal structure of K213S-PCK-ATP-Mg2+-Mn2+ demonstrates that Mn2+ is tetrahedrally coordinated in the active site, not octahedrally as occurred in other structures. By having Mn2+ in the tetrahedral coordination state, substrate binding in the active site of PCK is altered in a way that does not allow catalysis to occur.<p>The two crystal structures of <i>A. succiniciproducens</i> PCK were useful in quantifying the substrate-induced domain movement. A surface active site lid made up of residues 385 to 405 that had never been observed in any of the previous PCK crystal structures was observed in the <i>A. succiniciproducens</i> PCK-ATP-Mg2+-Mn2+-oxalate crystal structure. Mutational studies of this lid have shown it to be essential for the function of PCK; however, its exact function is not certain. It has been proposed that the lid has multiple functions. One is to sequester the substrates from bulk solvent. Another function may be to assist in domain closure. The third function may be to assist in the proper positioning of substrates in the active site.

La NADPH cytochrome P450 réductase (CPR) est une flavoprotéine multidomaines appartenant à la famille des diflavines réductases et un des composants essentiels du système redox des cytochromes P450. La CPR est formée de deux domaines catalytiques contenant des groupements prosthétiques FAD et FMN et d'un domaine de connexion. Le domaine FAD reçoit deux électrons du NADPH et les transfère un par un au domaine FMN, qui, à son tour, les transfère aux accepteurs. Le transfert d’électron du FMN vers les accepteurs nécessite un déplacement du domaine FMN par rapport au reste de la molécule. Au fils des années, les études structurales menées sur la CPR ont mis en évidence la réorganisation structurale et l’arrangement des domaines dans cette protéine. Cependant, les résultats de ces analyses ne fournissent pas d’informations concernant la vitesse à laquelle les mouvements des domaines de la CPR s’effectuent et n’incluent toujours pas les paramètres qui induisent le changement conformationnel ainsi que l'influence de ces changements sur l’activité catalytique de la CPR.Le projet de cette thèse a consisté à apporter de nouveaux éléments de compréhension sur la relation entre les changements conformationnels de la CPR et son cycle catalytique. La première partie de ce travail a porté sur le développement de stratégies de préparation au marquage des domaines catalytiques de la CPR, destinés à l’étude dynamique de cette protéine par le FRET. Différentes stratégies ont été envisagées parmi lesquelles l’incorporation de p-acétyle phénylalanine sur des positions définies dans la CPR. La deuxième partie de ce travail est consacrée à l’étude dynamique de la CPR via des techniques de RMN et SAXS combinées à des approches biochimiques. Les expériences menées ont permis de caractériser en solution et en absence de NaCl, la présence d’un état rigide, globulaire en conformation fermée dans laquelle les domaines FMN et FAD sont maintenus « verrouillés » par des interactions à l’interface entre ces deux domaines. L’augmentation de la concentration en NaCl permet une transition de cet état « verrouillé » vers un état plus ouvert pour lequel il n’y a plus d’interface entre les domaines FAD et FMN. L’état « déverrouillé » de la CPR correspond à un équilibre dynamique entre un ensemble de conformations en échange rapide. Cet équilibre est contrôlé par la force ionique et l’activité catalytique de la CPR est maximale lorsque les états verrouillés et déverrouillés sont également peuplés. Le modèle cinétique proposé par nos études a permis de mettre en évidence un lien direct entre la dynamique des domaines et l’activité du transfert d’électron au cours de cycle catalytique de la CPR. / NADPH cytochrome P450 reductase (CPR) is a multidomain flavoprotein that belongs to the diflavines reductase family. It is an essential component of redox system delivering electrons for cytochrome P450. CPR is composed of two catalytic domains containing FAD and FMN prosthetic groups and a connecting domain. FAD domain receives two electrons from NADPH and transfers them one by one to the FMN domain, which in turn transfers them to the acceptor. The electron transfer from FMN to the acceptor requires a large domain movement. Over the years, structural studies of CPR have highlighted the reorganization and arrangement of domains in this protein. However, the results of these analyses do not provide any information about how fast the domains movements takes place in CPR, and do not always include the parameters that induce conformational change as well as influence of those changes on the catalytic activity of the CPR. This thesis aims to bring new elements of comprehension on the relationship between conformational changes in CPR and its catalytic cycle. The first part of the work concerned the development of strategies to label the catalytic domains of CPR, a prerequisite for the dynamic study of this protein by FRET. Different strategies have been proposed including the incorporation of p-acetyl phenylalanine at defined positions in CPR. The second part of this work is devoted to the dynamic study of CPR through a combined SAXS, NMR and biochemical approaches. The experiments conducted allowed to characterize the presence of a rigid and globular state of closed conformation for CPR, in solution and in the absence of NaCl. In this conformation the FMN and FAD domains are kept "locked" by interface interactions between these two domains. Increasing the NaCl concentration permits the transition from the "locked" stats to an open conformation in which there is no more interface between the FAD and FMN domains. The "unlocked" state of CPR is a dynamic equilibrium between ensembles of conformations in fast exchange. This equilibrium is controlled by the ionic strength and CPR presents its maximum catalytic activity when the locked and unlocked states are equally populated. We proposed a kinetic model which allows demonstrating a direct link between the domain movement and electron transfer activity during the catalytic cycle of CPR.