Human pyruvate kinase performs the last step in glucose glycolysis in all cells and organisms and can be a key regulator of glycolytic flux. Pyruvate produced by PYK is transported into the mitochondria to fuel the TCA cycle, which enables the production of ATP; the main energy source of the cell. Human PYK contains four isoforms: M1 (found in muscle, heart and brain), M2 (in foetal cells and tumours), L (liver), and R (red blood cells) PYK. M2PYK plays a crucial role in tumour cell proliferation; by down-regulating metabolic flux, upstream metabolites can be used for protein and DNA synthesis. Reprogramming the metabolism of fast proliferating cells is called the 'Warburg effect'. The biological relevance of the different isoform activities is also discussed. For example RPYK in red blood cells is exposed to slowly altering metabolite concentrations, especially after intestinal absorption in plasma and RBCs uptake some of the metabolites. This thesis describes biochemical and biophysical studies of human M1PYK, M2PYK, LPYK, and RPYK. PYK is allosterically regulated by a range of metabolites. A comparative enzyme kinetics study of the four isoforms was performed to examine the mechanisms of activation and inhibition of these small molecule regulators, including all 20 amino acids and the thyroid hormone T3. The redox state of the environment was also found to be an important regulator of PYK activity. All four PYK isoforms were successfully expressed and purified. Interestingly, only M2PYK and RPYK were strongly regulated by amino acids and metabolites. We also found that the redox state regulates the activity of all four PYK isoforms as well as the sensitivity of M2PYK in response to natural regulators. These studies also confirmed the dissociation of tetrameric PYK into inactive monomers as an important mechanism of regulation, particularly for M2PYK activity. Nuclear magnetic resonance (NMR) and Small-angle X-ray scattering (SAXS) studies were performed to investigate the conformational behaviour of PYK isoforms in solution and to compare the effects of ligand binding. NMR data of all four isoforms reveal a conserved binding mechanism between isoforms and specific amino acids. SAXS data of all four isoforms demonstrate that ligands affect tetramerisation of PYK isoforms.

ABSTRACT Pyruvate kinase is an essential enzyme in the anaerobic glycolytic pathway of the erythrocyte. The clinical presentation of this enzyme deficiency is due to the haemolytic process that results from the inability of erythrocytes to generate sufficient ATP. Although pyruvate kinase and glucose-6-phosphate dehydrogenase deficiencies comprise more than 90% of all reported red cell enzyme disorders worldwide, the epidemiology of the disease in South Africa is unknown and there is no assay for pyruvate kinase activity currently being used in South Africa. This report describes the implementation of screening and quantitative assays for pyruvate kinase activity in the Red Cell Membrane Unit at the University of the Witwatersrand Medical School / NHLS. The accuracy, precision and reproducibility of the assay were verified. Furthermore, a patient with pyruvate kinase deficiency was confirmed and found to have 15% of normal enzyme activity at 37oC. The genetic abnormality was identified as a homozygous G1529A point mutation in exon 11 of the pyruvate kinase gene and to the candidate’s knowledge is the first mutation described in a South African kindred. The patient’s mother was heterozygous for the G1529A mutation and demonstrated an enzyme activity of 58% of normal at 37oC.

This thesis examines Escherichia coli pyruvate kinase type 1 (PK1), a regulatory enzyme core to energy metabolism. Specifically, this thesis characterises a series of mutations in PK1 that were found when populations of E. coli were evolved in a glucose-limited environment for 20,000 generations. The gene pykF, which codes for the PK1 enzyme, was found to have developed nonsynonymous mutations in all replicate populations. Although the mutations at the nucleotide level were not the same (i.e. not parallel), it is not clear whether parallel adaptation exists at the protein structure/function level. This study aimed to address this question by investigating the kinetic and biophysical properties of the wild-type and seven mutant enzymes. The recombinant wild-type PK1 enzyme used in this study was found to have steady state kinetics consistent with those previously reported. Unlike the rabbit kidney PK enzyme, E. coli PK1 was shown to have a very tight tetrameric structure (picomolar range), which was not affected by the enzyme’s substrates (PEP and ADP), or the allosteric effector (FBP), as judged by analytical ultracentrifugation with fluorescence detection. The mutated residues were highly conserved, and found to fall loosely into three groups: those at the active site (P70T, P70Q and D127N); those at the subunit interface (I264F, A301T and A301S); and at the allosteric binding site (G381A). The seven mutated PK1 enzymes were obtained by mutagenesis followed by protein purification. Steady state kinetic analysis showed that the mutated enzymes displayed a variety of functional changes, suggesting that the populations had not evolved in a parallel manner at the enzyme structure/function level. Mutations within the active site (P70T, P70Q and D127N) all showed a decrease in catalytic potency. P70 is located at the hinge connecting the A and B domains, which forms the active site. PK1-P70Q showed strong cooperative binding to PEP, similar to the wild-type enzyme, in the absence of FBP, whereas PK1-P70T had little cooperativity, suggesting changes in the active site. PK1-D127N showed severely attenuated activity, suggesting, for the first time, that this residue is essential for catalysis. Mutations at the subunit interface (I264F, A301T and A301S) all showed altered allosteric regulation, suggesting that this interface is important in the FBP allosteric response. PK1-I264F, which had lower activity, but greater affinity for PEP, displayed a decreased α-helix content (as judged by CD), indicating that a subunit interface helix that includes this residue had altered. Despite still having a similar response to FBP, PK1-G381A showed an increased affinity for PEP, which, together with an increased α-helix content, suggests that this mutation had changed the structure of the FBP binding domain. None of the mutated enzymes showed altered quaternary structure. Although the populations evolved parallel changes with respect to cell physiology, fitness, and gene expression, this study suggests, for the first time, that the populations have not evolved in a parallel way with respect to protein structure and function.

Many diseases correlate with abnormal glucose metabolism in cells and organisms. For instance, the human M2 isoform of the glycolytic enzyme pyruvate kinase (M2PYK) plays an important role in metabolic reprogramming of tumour cells whereby aerobic glycolysis or the ‘Warburg effect’ supports cell proliferation by accumulating necessary biomass. By contrast, gluconeogenesis may play an important role, as observed in certain types of trypanosomatid parasites (e.g. the amastigote form of Leishmania major) where anabolism is essential for infectious properties. Hence, these glucose metabolising enzymes are important potential drug targets for cancer and trypanosomiasis. However, many aspects of their regulatory mechanisms are still poorly understood. This thesis describes biochemical and structural studies on M2PYK and on L. major fructose-1,6-bisphosphatase (LmFBPase), providing insights into allosteric mechanisms and structure-based drug design for both enzymes. Human PYKs and LmFBPase were expressed and purified from Escherichia coli, and their kinetics were fully characterised. It was shown that certain amino acids regulate the activity of M2PYK allosterically, but in opposite ways, with some being inhibitors and others activators. X-ray crystallographic structures and biophysical data of M2PYK complexes with alanine, phenylalanine, serine or tryptophan reveal an R-/T-state oscillating model of M2PYK involving a 11° rotation of each subunit. In addition, M2PYK was demonstrated to be a redox-sensitive enzyme. Reducing reagents were shown to help maintain the tetramer and prevent its dissociation, and thereby to activate M2PYK, whereas oxidation and nitrosylation reagents functioned in the opposite sense. Nitrosylation assays showed that the main nitrosylated residue is Cys326 of M2PYK, which is located on the tetramer interface. Dynamics and modulator effects of PYKs were further studied by hydrogen–deuterium exchange by mass spectrometry. These observations highlight the important effects of amino acids on M2PYK regulation. M1PYK by contrast, was demonstrated to be a constitutively fully active pyruvate kinase, with minor effects from modulators. The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is a potential drug target against leishmaniasis. Here we present biochemical and structural studies for LmFBPase, by characterising its activity in a metal-dependent reaction, as well as its inhibition by AMP. The crystal structure of LmFBPase is a homotetramer, composed of monomers with alternating α/β/α/β/α ‘club sandwich’ topologies. In comparison with previously revealed LmFBPase structures, the AMP-complexed structure shows a rotated form of the tetramer. Comparisons of the structures reveal an ‘unlock-androtate’ allosteric mechanism in which AMP binding causes a series of structural changes culminating in an incomplete and non-productive active site. The structure of the effector site of LmFBPase shows a different conformation from human FBPases, thereby offering a potential specific target for Leishmania.

Glycolytic enzymes have been indicated as potential drug targets in trypanosomatid parasites such as Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania spp. Pyruvate kinase (PYK) catalyses the final reaction in the glycolytic pathway to produce ATP and pyruvate from ADP and phosphoenolpyruvate (PEP), and has been validated by RNAi experiments as a suitable drug target in T. brucei. This thesis describes biochemical and structural studies of PYKs from T. cruzi (TcPYK) and T. brucei (TbPYK), providing not only a foundation but also new clues for PYK-specific inhibitor screening and structure-based drug design. Soluble TcPYK and TbPYK (81% sequence identity) have been expressed and purified from E. coli, and their kinetics have been fully characterised. X-ray crystal structures of apoenzyme TcPYK (apo TcPYK), and of TbPYK in complex with fructose 2,6-bisphosphate (F26BP) (TbPYK/F26BP/Mg) have been determined, and each possesses a tetrameric architecture composed of four identical protein chains. Each chain contains four domains which are A-domain, B-domain, C-domain and N-terminal domain. The active site is located in the cleft between the A- and B-domains, while the F26BP-bound effector site is within the C-domain. The conformational transition between inactive T-state and active R-state for both enzymes requires a concerted 8o rigid-body rotation of each of the four AC-cores (Aand C-domains) in the tetramer. During the T- to R-state transition induced by F26BP binding, the side chain of Arg311 is re-orientated to stabilise the short Aα6′ helix at the active site, and the flexible loop at the effector site is stabilised by F26BP. In this active conformation additional salt bridges form across the C-C interface to lock the enzyme in a more stable R-state. TbPYK/F26BP/Mg is the first ‘effector only’ PYK structure and identifies a third Mg2+ binding site (Mg-3) which is distinct from the two canonical Mg2+ binding sites. The substrate PEP was soaked into crystals of TbPYK/F26BP/Mg resulting in an ‘in crystallo’ 23° B-domain rotation forming a partially closed active site. This is accompanied by active site side-chain reorientations, and the movement of Mg2+ from its ‘priming’ position Mg-3 to its canonical position Mg-1. It is plausible that Mg2+ is retained in its ‘priming’ position after product release to act as a co-activator with F26BP to maintain the enzyme in its R-state conformation, as long as F26BP is present. The inherent oxaloacetate decarboxylase activity of PYK was reported over 30 years ago and has been further characterised by 1H NMR studies in this thesis. In addition, a series of TbPYK structures in complex with product (pyruvate), with analogues of the decarboxylase substrate oxaloacetate (D-malate and α-ketoglutarate), or with the competitive inhibitor oxalate have been determined by crystal soaking, and indicate that both decarboxylase activity and kinase activity share a common active site. A proposed mechanism explains the conserved decarboxylase activity of PYK where the active-site Mg2+ and Lys239 in TbPYK (which is conserved between species) play essential roles in the decarboxylation reaction. Three strategies for designing novel inhibitors against trypanosomatid PYKs have been proposed in this thesis. (1) Develop selective modulators to increase the binding affinity of inhibitors. As an example, F16BP has been shown to regulate the inhibitory effect of PEP analogues (oxalate, D-malate, α-ketoglutarate, malonate and L-tartrate) on TbPYK activity. (2) Develop allosteric inhibitors in order to lock trypanosomatid PYKs in an inactive state where the enzyme has low affinity for substrate binding. (3) A third strategy is to combine multiple modulators and inhibitors to increase the inhibition efficiency and selectivity.

Pyruvate kinase M2 (PKM2) has been studied for decades on its role in cancer metabolism. Recently, PKM2 is highlighted again for its new function: promoting gene transcription by acting as a protein kinase. Moreover, the PKM2 levels in patient circulation have been used as a diagnostic marker for many types of cancers. However, it remains unclear whether PKM2 in blood circulation has any physiological or pathological function. In my dissertation, I demonstrate that PKM2 released from cancer cells facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes endothelial cell proliferation, migration and survival. Only the dimeric PKM2, not the tetrameric PKM2 possesses the activity in angiogenesis promotion. Our results further indicate that PKM2 regulates angiogenesis by integrin αvβ3 activation and integrin redistribution. I also found that PKM2 enhances drug resistance of cancer cells expressing integrin αvβ3.

There is a growing body of interest to understand the regulation of allosteric proteins. Allostery is a phenomenon of protein regulation whereby binding of an effector molecule at a remote site affects binding and activity at the protein‟s active site. Over the years, these sites have become popular drug targets as they provide advantages in terms of selectivity and saturability. Both experimental and computational methods are being used to study and identify allosteric sites. Although experimental methods provide us with detailed structures and have been relatively successful in identifying these sites, they are subject to time and cost limitations. In the present dissertation, Molecular Dynamics Simulations (MDS) and Principal Component Analysis (PCA) have been employed to enhance our understanding ofallostery and protein dynamics. MD simulations generated trajectories which were then qualitatively assessed using PCA. Both of these techniques were applied to two important trypanosomatid drug targets and controlling enzymes of the glycolytic pathway - pyruvate kinase (PYK) and phosphofructokinase (PFK). Molecular Dynamics simulations were first carried out on both the effector bound and unbound forms of the proteins. This provided a framework for direct comparison and inspection of the conformational changes at the atomic level. Following MD simulations, PCA was run to further analyse the motions. The principal components thus captured are in quantitative agreement with the previously published experimental data which increased our confidence in the reliability of our simulations. Also, the binding of FBP affects the allosteric mechanism of PYK in a very interesting way. The inspection of the vibrational modes reveals interesting patterns in the movement of the subunits which differ from the conventional symmetrical pattern. Also, lowering of B-factors on effector binding provides evidence that the effector is not only locking the R-state but is also acting as a general heat-sink to cool down the whole tetramer. This observation suggests that protein rigidity and intrinsic heat capacity are important factors in stabilizing allosteric proteins. Thus, this work also provides new and promising insights into the classical Monod-Wyman-Changeux model of allostery.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This study aimed to assess the contribution of anaplerotic C prOVISIOn VIa phosphoenolpyruvate carboxylase (pEPc, EC 4.1.1.31), during Pi stress in the root and nodule components of Lupinus angustifolius. The role of PEPc in DIC metabolism in roots and nodules of phosphate-starved plants was studied. The symbioses involving leguminous plants and species of Rhizobium and Bradyrhizobium bacteria form an integral part of effective management ofN in the environment. In agricultural settings, roughly 80% of this biologically fixed N2 comes from this type of symbiotic relationship. Nitrogen-fixing bacteria in concert with legumes fix atmospheric nitrogen, which is then available to the infected plant. Worldwide, legumes are grown on approximately 250 Mha and they fix about 90 Tg (90 billion tons) of N, per year. The overall stoichiometry for nitrogen assimilation in the nodule requires one molecule of oxaloacetate to be converted to one molecule of asparagine per dinitrogen molecule fixed. One possible source for the required oxaloacetate is the reaction catalysed by PEPc. The reaction catalysed by PEPc is a major source of anaplerotic carbon for the plant and it is expected that this reaction will be even more important to plants under Pi stress, as the reaction is not ATP-dependent. Seeds of Lupinus angustifolius (cv. Wong a) were inoculated with Rhizobium sp. (Lupinus) bacteria and grown in hydroponic culture. Tanks were supplied with either 2 11MP04 (LP) or 2 mM P04 (HP) and air containing 360 ppm CO2. Roots experienced pronounced P stress with a greater decline in Pi, compared to nodules. Under P stress, PEPc activities increased in roots but not in nodules and these changes were not related to the expression of the enzyme. Root and nodular PEPc were not regulated by expression, but possibly by posttranslational control. LP roots also synthesised more pyruvate from malate than LP nodules. The role of pyruvate accumulation under Pi stress, was further highlighted by the metabolism of PEP via both the pyruvate kinase (PK, Ee 2.7.1.40) and PEPc routes. The enhanced PK activities supported these high pyruvate levels. The results show unequivocally that nodules do not experience P stress to the same extent as roots. Implications of the findings are that nodules require low P to function normally. Maintenance of phosphate levels in nodules may be at the expense of host. It can be suggested that when nodules are P-starved they can become aggressive scavengers for available P and even out-compete roots. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die bydrae van anaplerotiese koolstof-voorsiening via fosfo-enolpirovaatkarboksilase (pEPc, EC 4.1.1.31), tydens fosfaatstremming in die wortels en wortelknoppies van Lupinus angustifolius te bepaal. Die rol van PEPc in die metabolisme van opgeloste anorganiese koolstofdioksied in fosfaat-beperkte wortels en wortelknoppies is ondersoek. Die simbiose tussen peulplante en spesies van Rhizobium en Bradyrhizobium bakterieë vorm 'n integrale deel van die doeltreffende bestuur van stikstof in die omgewing. In die landbou word ongever 80 %van biologies-gefikseerde stikstof deur hierde simbiotiese verhouding geproduseer. Stikstotbindende bakterieë, in simbiose met peulplante, fikseer atmosferiese stikstof, wat dan beskikbaar is vir die geïnfekteerde plant. Wêreldwyd fikseer peulplante ongeveer 90 biljoen ton stikstof per jaar. Die algehele stoïgiometrie vir stikstof-fiksering in wortelknoppies vereis dat een molekule oksaalsuur na een molekule asparagien omgesit word per stikstofmolekule wat gefikseer word. Een moontlike bron vir die benodigde oksaalsuur is die reaksie wat deur PEPc gekataliseer word. Die reaksie wat deur PEPc gekataliseer word is 'n belangrike bron van anaplerotiese koolstof vir die plant en dit word vermoed dat hierdie reaksie van nog groter belang sal wees vir plante onder fosfaatstremming, omdat die reaksie nie ATP-afhanklik is nie. Sade van Lupinus angustifolius (cv. Wonga) is geïnokuleer met Rhizobium sp. (Lupinus) bakterieë en gekweek in waterkultuur. Tenke is voorsien met óf 2 !lM P04 (LP), óf 2 mM P04 (HP) en lug wat 360 ppm CO2 bevat het. Wortels het skerp fosfaatstremming ervaar, met 'n groter afname in Pi, vergelykbaar met wortelknoppies. Tydens fosfaatstremming het die aktiwiteit van PEPc toegeneem in wortels, maar nie in wortelknoppies nie en hierdie veranderinge was nie verwant aan die uitdrukking van die ensiem nie. PEPc van wortels en wortelknoppies is nie gereguleer deur uitdrukking nie, maar moontlik deur post-translasie kontrole. Wortels onder 'n lae-fosfaat voorsiening het ook meer pirodruiwesuur vanaf malaat gesintetiseer as wortelknoppies. Die rol van pirodruiwesuur-akkumulering tydens fosfaatstremming is verder beklemtoon deur die metabolisme van PEP via beide die pirovaatkinase- (PK, EC 2.7.1.40) en PEPc- roetes. Die verhoogde PK-aktiwiteite verklaar hierdie hoër vlakke van pirodruiwesuur. Die resultate toon ondubbelsinnig dat wortelknoppies me tot dieselfde mate fosfaatstremming ervaar as wortels nie. Dit impliseer dat wortelknoppies min fosfaat benodig om normal te funksioneer. Handhawing van fosfaatvlakke in wortelknoppies mag ten koste van die wortel wees. Dit is moontlik dat, wanneer wortelknoppies fosfaatbeperk is, hulle aggressiewe opruimers word vir beskikbare fosfaat en selfs beter funksioneer as die wortels.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The influence of body size on metabolic rate, muscle enzyme activities, and the underlying patterns of mRNA for these enzymes were explored in an effort to explain the genetic basis of allometric variation in metabolic enzymes. Two pairs of sister species of centrarchid fishes were studied: black bass (largemouth bass, Micropterus salmoides and smallmouth bass, Micropterus dolomieui), and sunfish (pumpkinseed, Lepomis gibbosus and bluegill, Lepomis macrochirus). The goal was to assess the regulatory basis of both intraspecific and interspecific variation in relation to body size, as well as gain insights into the evolutionary constraints within lineages. Whole animal routine metabolic rate showed scaling coefficients not significantly different from 1, ranging from +0.87 to +0.96. However, there were significant effects of body size on the specific activities of oxidative and glycolytic enzymes. Mass-specific activity of the oxidative enzyme citrate synthase (CS) scaled negatively with body size in each species, with scaling coefficients ranging from -0.15 to -0.19 whereas the glycolytic enzyme pyruvate kinase (PK) showed positive scaling, with scaling coefficients ranging from +0.08 to +0.23. The ratio of mass-specific enzyme activity in PK to CS increased with body size, whereas the ratio of mRNA transcripts of PK to CS was unaffected, suggesting the enzyme relationships were not due simply to transcriptional regulation of both genes. The mass-dependent differences in PK activities were best explained by transcriptional regulation of the muscle PK gene; PK mRNA was a good predictor of PK specific enzyme activity within species and between species. Conversely, CS mRNA did not correlate with CS specific enzyme activities, suggesting post-transcriptional mechanisms may explain the observed inter-specific and intraspecific differences in oxidative enzymes. / Thesis (Master, Biology) -- Queen's University, 2007-10-31 11:55:28.757