Lys169 of Human Glucokinase Is a Determinant for Glucose Phosphorylation: Implication for the Atomic Mechanism of Glucokinase Catalysis

Glucokinase (GK), a glucose sensor, maintains plasma glucose homeostasis via phosphorylation of glucose and is a potential therapeutic target for treating maturity-onset diabetes of the young (MODY) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To characterize the catalytic mechanism of glucose phosphorylation by GK, we combined molecular modeling, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, experimental mutagenesis and enzymatic kinetic analysis on both wild-type and mutated GK. Our three-dimensional (3D) model of the GK-Mg2+-ATP-glucose (GMAG) complex, is in agreement with a large number of mutagenesis data, and elucidates atomic information of the catalytic site in GK for glucose phosphorylation. A 10-ns MD simulation of the GMAG complex revealed that Lys169 plays a dominant role in glucose phosphorylation. This prediction was verified by experimental mutagenesis of GK (K169A) and enzymatic kinetic analyses of glucose phosphorylation. QM/MM calculations were further used to study the role of Lys169 in the catalytic mechanism of the glucose phosphorylation and we found that Lys169 enhances the binding of GK with both ATP and glucose by serving as a bridge between ATP and glucose. More importantly, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. Our findings provide mechanistic details of glucose phorphorylation catalyzed by GK, and are important for understanding the pathogenic mechanism of MODY

Computational chemistry studies of subtypes B and South African C HIV proteases.

Master of Medical Science in Pharmacy. University of KwaZulu-Natal, Durban, 2016.HIV/AIDs is a prevalent disease infecting millions of people throughout the world. Although a lot of improvement has been achieved over the year in regard to the reduction of AIDs related deaths, a huge task lies ahead as the HIV/AIDs global epidemic keeps spreading annually. It is therefore paramount to discover and develop more and efficient drug inhibitors against HIV. The HIV protease (HIV PR) is a C2-symmentric homodimer and consisting of 99-amino acids in each monomer and because of the important role it plays in the HIV mutation, it became a major HIV drug target for the past three decades. It is on this basis that various effective antiretroviral protease inhibitors have been designed and approved for application in HIV therapy.The HIV subtype B strain is prominent in Europe and North America and is the most researched virus. The majority of the antiretroviral drugs were designed and tested against HIV subtype B. However, non-subtype B strains of the HIV virus makes up most of these infections in Southern and Eastern Africa, which are highly affected regions in the world. In South Africa, subtype C HIV-1 is the dominant strain and little research has been done regarding drug design for this subtype or testing of the effectiveness of the HIV approved antiretroviral drugs against these non-subtype B strains. Two potentially devastating mutations of subtype C-SA HIV PR were recently reported by our group. These were designated I36T↑T and L38L↑N↑L HIV PR. The I36T↑T PR mutant includes an extra amino acid, the mutation occurs at position 36 (isoleucine to threonine) and is followed by an insertion at the second threonine indicated by the upward arrow. The L38L↑N↑L PR mutant involves two amino acids insertions that is completely different from the usual 99-amino acids HIV PR, as well as five point mutations occur at the E35D, I36G, N37S, M46L and D60E. The two insertions occur at position 38 (asparagine and leucine) indicated by the two upward arrows. Therefore, the I36T↑T and L38L↑N↑L mutations consist of 100 and 101-amino acids in each monomer of the proteases respectively.In this thesis, a hybrid computational model (QM: MM) using the ONIOM approach was followed. The selected FDA inhibitors were complexed with the various proteases in the active pocket interacting with Asp 25/25' catalytic residues using the same pose in the subtype B PR as a reference X-ray structure. The HIV PR inhibitors and Asp 25/25' were treated at a high-level with quantum mechanics (QM) theory using B3LYP/6-31G(d), and the remaining HIV PR residues were considered at a low layer using molecular mechanics (MM) with the AMBER force field. This method was applied to calculate the binding free interaction energies of the selected FDA approved HIV PR drugs complexed to the HIV protease enzyme. The aim was to create and test this computational model that will reflect the experimental binding energies against subtype B, C-SA HIV PR and also a mutant from the subtype C-SA PR designated L38L↑N↑L HIV PR. The calculated binding free interaction energies results from the subtype B follow a satisfactory trend with the experimental data. However, the C-SA HIV PR inhibitor―enzyme complexes showed some discrepancies and this was ascribed to the simplified computational model that omitted water in the active site of the enzyme. The calculated binding free interaction energies for L38L↑N↑L PR as well as experimental results, showed reduced binding affinities for all the selected FDA approved inhibitors in comparison with the subtype C-SA HIV PR. The deviation could be as a result of the insertion and mutation of the subtype C HIV-1 PR that is expected to have a significant effect in altering either the binding affinity of the HIV PR inhibitors and or characteristics of the parent protease. The computational model used in this research will be improved by introducing water into the active pocket of the Asp 25/25' catalytic residues that will be treated at least at semi-empirical level. Optimization of the different ONIOM levels will be attempted in order to accurately predict activities of new potential HIV PR inhibitors

Catalytic Mechanism Investigation of Lysine-Specific Demethylase 1 (LSD1): A Computational Study

Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, is a flavin-dependent amine oxidase which specifically demethylates mono- or dimethylated H3K4 and H3K9 via a redox process. It participates in a broad spectrum of biological processes and is of high importance in cell proliferation, adipogenesis, spermatogenesis, chromosome segregation and embryonic development. To date, as a potential drug target for discovering anti-tumor drugs, the medical significance of LSD1 has been greatly appreciated. However, the catalytic mechanism for the rate-limiting reductive half-reaction in demethylation remains controversial. By employing a combined computational approach including molecular modeling, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations, the catalytic mechanism of dimethylated H3K4 demethylation by LSD1 was characterized in details. The three-dimensional (3D) model of the complex was composed of LSD1, CoREST, and histone substrate. A 30-ns MD simulation of the model highlights the pivotal role of the conserved Tyr761 and lysine-water-flavin motif in properly orienting flavin adenine dinucleotide (FAD) with respect to substrate. The synergy of the two factors effectively stabilizes the catalytic environment and facilitated the demethylation reaction. On the basis of the reasonable consistence between simulation results and available mutagenesis data, QM/MM strategy was further employed to probe the catalytic mechanism of the reductive half-reaction in demethylation. The characteristics of the demethylation pathway determined by the potential energy surface and charge distribution analysis indicates that this reaction belongs to the direct hydride transfer mechanism. Our study provides insights into the LSD1 mechanism of reductive half-reaction in demethylation and has important implications for the discovery of regulators against LSD1 enzymes

A Multi-Scale Computational Study on the Mechanism of Streptococcus pneumoniae Nicotinamidase (SpNic)

Nicotinamidase (Nic) is a key zinc-dependent enzyme in NAD metabolism that catalyzes the hydrolysis of nicotinamide to give nicotinic acid. A multi-scale computational approach has been used to investigate the catalytic mechanism, substrate binding and roles of active site residues of Nic from Streptococcus pneumoniae (SpNic). In particular, density functional theory (DFT), molecular dynamics (MD) and ONIOM quantum mechanics/molecular mechanics (QM/MM) methods have been employed. The overall mechanism occurs in two stages: (i) formation of a thioester enzyme-intermediate (IC2) and (ii) hydrolysis of the thioester bond to give the products. The polar protein environment has a significant effect in stabilizing reaction intermediates and in particular transition states. As a result, both stages effectively occur in one step with Stage 1, formation of IC2, being rate limiting barrier with a cost of 53.5 kJ•mol−1 with respect to the reactant complex, RC. The effects of dispersion interactions on the overall mechanism were also considered but were generally calculated to have less significant effects with the overall mechanism being unchanged. In addition, the active site lysyl (Lys103) is concluded to likely play a role in stabilizing the thiolate of Cys136 during the reaction

NEW COMPUTATIONAL METHODS AND ALGORITHMS FOR SEMICONDUCTOR SCIENCE AND NANOTECHNOLOGY

Thesis (Ph.D.) - Indiana University, Chemistry, 2015The design and implementation of sophisticated computational methods and algorithms are critical to solve problems in nanotechnology and semiconductor science. Two key methods will be described to overcome challenges in contemporary surface science. The first method will focus on accurately cancelling interactions in a molecular system, such as modeling adsorbates on periodic surfaces at low coverages, a problem for which current methodologies are computationally inefficient. The second method pertains to the accurate calculation of core-ionization energies through X-ray photoelectron spectroscopy. The development can provide assignment of peaks in X-ray photoelectron spectra, which can determine the chemical composition and bonding environment of surface species. Finally, illustrative surface-adsorbate and gas-phase studies using the developed methods will also be featured

Computational studies of mycobacterium tuberculosis L, d-transpeptidase2.

Masters Degree. University of KwaZulu-Natal, Durban.Tuberculosis (TB) is still one of the most highly elusive lethal transmittable diseases to eradicate and persist to be a major threat to public health due to emergence of drug resistance. Drug-resistant is steadily increasing worldwide, therefore, there is an urgent need for development of improved efficacious antibiotics and novel drug targets to successfully contain the disease. Peptidoglycan layer (PG) is the major attribute in bacterial cell envelope and is essential for protection and growth in all bacterial species including Mycobacterium tuberculosis(Mtb). The biosynthesis pathway for PG is extremely intricate and involves numerous interconnected metabolites such as N-acetylmuramic(NAM) acid and N-acetylglucosamine(NAG), that are required during transpeptidation. These two sugar molecules are linked together by a β (1-4) glycosidic bond and the NAM attaches 3-5 amino acid peptide stems. Consequently, the peptidoglycan strands are cross linked by transpeptidases, namely D, D- and L, D-transpeptidases, forming crucial 4→3 and 3→3 cross-linkages respectively. Both D, D- and L, D-transpeptidases need to be inhibited concomitantly to eradicate the bacterium. L, D-transpeptidase 2 (LdtMt2) is one of the paralogs that is essential for Mtb growth, cell morphology and virulence during the chronic stage of the disease. This paralog has major influence in drug resistance and persistence of tuberculosis. The traditional β-lactam family of antibiotics have been reported to be effective against Mtb following the inactivation of β-lactamases (BlaC) known to rapidly hydrolyze the core β-lactam ring. The classic penicillins inhibit D, D-transpeptidases, while L, D transpeptidases are blocked by carbapenems. Despite several studies in this field, to the best of our knowledge, limited attention has been paid to the inhibition mechanism of LdtMt2 using carbapenem derivatives. In this regard, we need to explore reliable alternative strategies that are most cost-effective in terms of investigating the interactions of FDA approved carbapenems against Mtb L, D-Transpeptidases and study the role of explicit water molecule confined in the active site. As a result, computational chemistry has provided the possibility to sightsee and investigate this problem with relatively cost effective computational techniques. In this thesis, we applied a hybrid quantum mechanics and molecular mechanics techniques (QM:MM), Our own N-layered Integrated molecular Orbital and Molecular Mechanics (ONIOM) approach, to investigate the binding interaction energies of carbapenems (biapenem, imipenem, meropenem and tebipenem) against L, D-transpeptidase 2. Furthermore, the role of explicit water molecule confined in the active site was also explored using the same hybrid method to ascertain the nature of binding interaction energies of carbapenems against LdtMt2. In all the investigated carbapenem─LdtMt2 complexes, the carbapenems and the catalytic active site residues of LdtMt2 (Cys205, His187, Ser188, His203 and Asn207) were treated at QM (B3LYP/6-31+G(d)) level of theory whereas the remaining part of the complexes were treated at MM level (AMBER force field). The explicit water molecules near the carbapenems were considered and treated at QM as well. The obtained findings of Gibbs free energy (G), enthalpy (H) and entropy (S) for all studied complexes showed that the carbapenems exhibit reasonable binding interactions towards LdtMt2. This can be attributed by the structural dissimilarities of the carbapenems side chain which significantly induce conformational changes in the LdtMt2. In comparison, the binding free energy calculations of the model system with explicit water molecule yielded significant binding interaction energies. The QTAIM and NBO results confirmed the nature of binding free energies that the topological properties of atoms in molecules and the delocalization of electrons are from a bonding to antibonding orbitals in hydrogen bond interactions and this has enhanced the stability of carbapenem―LdtMt2 complexes. We believe that molecular insight of the carbapenems binding to LdtMt2 and the role of explicit solvent will enable us to understand the inhibition mechanisms

Developments in multiscale ONIOM and fragment methods for complex chemical systems

Multiskalenprobleme werden in der Computerchemie immer allgegenwärtiger und bestimmte Klassen solcher Probleme entziehen sich einer effizienten Beschreibung mit den verfügbaren Berechnungsansätzen. In dieser Arbeit wurden effiziente Erweiterungen der Multilayer-Methode ONIOM und von Fragmentmethoden als Lösungsansätze für derartige Probleme entwickelt. Dabei wurde die Kombination von ONIOM und Fragmentmethoden im Rahmen der Multi-Centre Generalised ONIOM entwickelt sowie die eine Multilayer-Variante der Fragment Combinatio Ranges. Außerdem wurden Schemata für elektronische Einbettung derartiger Multilayer-Systeme entwickelt. Der zweite Teil der Arbeit beschreibt die Implementierung im Haskell-Programm "Spicy" und demonstriert Anwendungen derartiger Multiskalen-Methoden

Theoretical studies on the reaction mechanisms of catalytic organic reactions

Master'sMASTER OF SCIENC