<div> <div> <div> <p>Currently there is a need for research that explores students’ understanding of advanced topics in order to improve teaching and learning beyond the context of introductory-level courses. This work investigates students’ reasoning about graphs used in enzyme kinetics. Using semi-structured interviews and a think aloud-protocol, 14 second-year students enrolled in a biochemistry course were provided two graphs to prompt their reasoning, a typical Michaelis-Menten graph and a Michaelis-Menten graph involving enzyme inhibition. Student responses were coded using a combination of inductive and deductive analysis, influenced by the resource-based model of cognition. Results involve a discussion regarding how students utilized mathematical resources to reason about chemical kinetics and enzyme kinetics, such as engaging in the use of symbolic/graphical forms and focusing on surface-level features of the equations/graphs. This work also addresses student conceptions of the particulate-level mechanism associated with competitive, noncompetitive, and uncompetitive enzyme inhibition. Based on the findings of this study, suggestions are made regarding the teaching and learning of enzyme kinetics. </p> </div> </div> </div>

Dynamic structural fluctuations occurring over a broad range of timescales are now known to facilitate the catalytic function of enzymes, but there is less comprehensive experimental evidence linking fast-timescale, high frequency motions to the reaction coordinate. Interest in the role of such motions has recently surged and been the subject of intensive experimental efforts, in part due to the identification of enzymatic hydride tunnelling reactions. This mechanism involves transiently degenerate product and reactant states, which enable H-transfer to occur instantaneously without the need to surmount the activation barrier associated with traditional transition-state based models of enzyme catalysis. The primary gauge of tunnelling in enzyme-catalysed reactions is the identification of temperature dependent kinetic isotope effects (KIEs), i.e. the relative rates of a reaction where the transferred atom is substituted for an alternate isotope. The identification of temperature-, and also pressure-, dependent KIEs has resulted in the emergence of new models of describing enzymatic H-transfer. These invoke a role for fast-timescale protein motions that 'promote' transfer via tunnelling. A popular model system for studying enzymatic H-tunnelling reactions is Pentaerythritol tetranitrate reductase, which belongs to the Old Yellow Enzyme (OYE) family of ene-reductases. These nicotinamide coenzyme dependent oxidoreductases catalyse the stereospecific reduction of alpha/β-unsaturated alkene containing substrates. Here, the importance of donor-acceptor distances in determining the observed rate of PETNR reduction with NAD(P)H is probed via a detailed structural and kinetic analysis of site-directed variants. In addition, an investigation of distance-dependent Nuclear Overhauser effects via Nuclear Magnetic Resonance (NMR) spectroscopy is undertaken to assess active site organisation and measure donor-acceptor distances in PETNR-substrate complexes. A variable pressure NMR study reveals how NOE build- up is perturbed in high-energy conformers favoured as a result of the application of increased hydrostatic pressures. Recently there has been interest in exploiting the stereoselective properties of reactions catalysed by ene-reductase enzymes for use in biocatalytic reactions to produce industrially valuable compounds from renewable sources. The reactions of PETNR and additional OYE enzymes, Thermophilic old yellow enzyme and Xenobiotic reductase A, with both natural coenzymes and a set of synthetic Nicotinamide Coenzyme Biomimetics (NCBs) are also characterised. The NCBs represent affordable and fast-reacting alternatives to the physiological coenzymes. Reactions with NCBS are also shown to proceed via a tunnelling mechanism and furthermore, that enhanced donor-acceptor sampling correlates with the faster reactivity seen with these compounds.

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

Conjugates of soybean trypsin inhibitor (SBTI) and potato serine protease inhibitor (PSPI) immobilized on metal oxide particles of ~100nm diameter were prepared. Inhibition of trypsin hydrolysis of BAPA by these conjugates was measured and enzyme kinetics constants kcat, KM, kcat/KM and ki were determined. Metal oxide particles presented an inhibitory effect similar to that of a competitive inhibitor, noticed through the increase value of the K M constant. Furthermore, PSPI conjugates had the highest inhibition of trypsin, illustrated by the significantly higher value of KM relative to the value for particles only.

Transglutaminases are a family of enzymes expressed in various tissues of our body. Some are expressed ubiquitously while others are specific to a tissue. Their primary catalytic activity is to crosslink substrates via an isopeptidic bond. The work described in this thesis focuses on two of these transglutaminases; human tissue transglutaminase (hTG2) and human factor XIIIa (FXIIIa). Divided into two projects for each enzyme, the main objective of this thesis was directed towards the discovery of potent and selective covalent inhibitors for each isozyme, namely hTG2 and hFXIIIa. The first project was concentrated on the inhibition of hTG2 activity. Ubiquitously expressed in tissues, hTG2 is a multifunctional enzyme. Its primary activity is the formation of isopeptide bonds between glutamine and lysine residues found on the surface of proteins or substrates. In addition to its catalytic activity, hTG2 is also a G-protein, distinguishing it from other members of the transglutaminase family. Much evidence illustrates that hTG2’s multifunctional abilities are conformationally regulated between its “open” and “closed” forms. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases; however, most evidence has been collected for its association with fibrosis and celiac sprue. More recently, elevated hTG2 expression has been linked to cancer stem cell survival and metastatic phenotype in certain cancer cells. These findings call for the development of suitable and potent inhibitors that selectivity inactivate human hTG2 as a potential therapeutic target. Starting with previously designed acrylamide based peptidomimetic irreversible inhibitors, a structure-activity relationship (SAR) study was conducted. In this work, >20 novel irreversible inhibitors were prepared and kinetically evaluated. Our lead inhibitors allosterically inhibited GTP binding by locking the enzyme in its open conformation, as demonstrated both in vitro and in cells. Furthermore, our most potent and efficient irreversible inhibitors revealed selectivity for hTG2 over other relevant members of the transglutaminase family (hTG1, hTG3, hTG6 and hFXIIIa), providing higher confidence towards our goal of developing an ideal drug candidate. The second project was concentrated on the inhibition of hFXIIIa activity. In the blood, coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A subunits (FXIII-A2) and two carrier/inhibitory B (FXIII-B2) subunits. It is a zymogen, which is converted into active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin proteolytic activity and Ca2+ binding. hFXIII is essential for hemostasis and thus its deficiency results in severe bleeding conditions. Further, hFXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Due to the enzyme’s involvement in the stability of blood clots, inhibition of hFXIIIa activity has been linked to thrombotic diseases. Furthermore, inhibitors of the enzyme have the therapeutic potential to be used as anticoagulant agents. The current number of selective and potent inhibitors of hFXIIIa are few, mainly due to the similarity between its catalytic pockets and hTG2. Inspired by a poorly reactive hTG2 inhibitor discovered in this work’s hTG2 SAR study, we synthesized a small library of covalent inhibitors for hFXIIIa. Our kinetic results from this pioneering SAR study will pave the way for future hFXIIIa inhibitor SAR studies.

<p>The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using <i>Pichia pastoris</i> was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the K_M^app is more than 10² higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.</p>

The protein degradation process is of vital importance for the cell to maintain cellular functions. An important enzyme in this process is the multimeric tripeptidyl-peptidase II (TPP II). It removes tripeptides from a free N-terminus of the substrates. TPP II has broad substrate specificity and wide-spread distribution, suggesting that the TPP II gene is a house-keeping gene. However, the levels of both mRNA and TPP II protein varies during different conditions and the TPP II gene promoter was therefore identified and characterized. It is a 215 bp fragment just upstream of the coding sequence. This fragment lacks a TATA-box but contains an initiator, two inverted CCAAT-boxes and an E-box. The CCAAT-boxes and the E-box were found to bind the nuclear factor Y (NF-Y) and upstream stimulatory factor-1 (USF-1) respectively. The CCAAT-boxes appear to be most important for the transcriptional activation. Furthermore, several silencer element were identified further upstream of the 215 bp promoter and the octamer binding factor Oct-1 was found to bind one of these fragments. If Oct-1 is responsible for the inhibition of the transcription of the TPP II gene remains to be investigated. In addition, the substrate specificity was investigated. For this purpose an expression system using Pichia pastoris was developed. The purified recombinant TPP II was found to have the same enzymatic properties as the native enzyme. In order to identify the amino acids involved in the binding of the N-terminus of the substrate, wild-type murine TPP II and four mutants E305Q, E305K, E331Q and E331K were purified. Steady-state kinetic analysis clearly demonstrated that both Glu-305 and Glu-331 are important for this binding as the KMapp is more than 102 higher for the mutants than wild-type. Finally, the pH-dependence for cleavage of two chromogenic substrates was compared for TPP II from different species.

Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

Significant advancements in the field of membrane protein crystallography have provided in recent years invaluable images of transporter structures. These structures, however, are static and require complementary kinetic insight to understand how their mechanisms work. Electrophysiological studies of transporters permit the high quality kinetic measurements desired, but there are significant difficulties involved in analyzing and interpreting the data. Current methods allow a variety of kinetic parameters to be measured but there is a disconnect between these parameters and a fundamental understanding of the carrier. The intent of this research was to contribute new tools for studying the electrogenic kinetics of membrane transport proteins, to understand the link between these kinetics and the carrier, and to ultimately understand the mechanisms involved in transport. In this vein, two projects are explored covering two important kinetic time domains, transient and steady-state. The transient project studies the conformational changes of the unloaded carrier of SGLT1 through a multi-exponential analysis of the transient currents. Crystal structures have potentially identified a gated rocker-switch mechanism and the transient kinetics are used to support and study this kinetically. A protocol taking advantage of multiple holding potentials is used to measure the decay time constants and charge movements for voltage jumps from both hyperpolarizing and depolarizing directions. These directional measurements provide insight into the arrangement of the observed transitions through directional inequalities in charge movement, by considering the potential for a slow transition to hide a faster one. Ultimately, four carrier decays are observed that align with the gated rocker-switch mechanism and can be associated one-to-one with the movement of a gate and pore on each side of the membrane. The steady-state project considers a general theoretical model of transporter cycling. Recursive patterns are identified in the steady-state velocity equation that lead to a broad understanding of its geometric properties as a function of voltage and substrate concentration. This results in a simple phenomenological method for characterizing the I–V curves and for measuring the kinetics of rate limiting patterns in the loop, which we find are the basic structures revealed by the steady-state velocity.

2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl compounds and nitrite, with oxygen as the electron acceptor. Although nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in chemical industry for a quick and effective way of synthesizing common reagents. Consequently, the biochemical and biophysical analysis of 2-nitropropane dioxyganase has a potential for bioremediation purposes. In this study, recombinant enzyme is purified to high levels, allowing for detailed characterization. The biochemical analysis of 2-nitropropane dioxygenase presented in this study has established that enzyme utilizes alkyl nitronates as substrates by forming an anionic flavosemiquinone in catalysis. The enzyme is inhibited by halide ions, does not contain iron and has a positive charge located close to the N(1)-C(2)=O locus of the isoalloxazine moiety of the FMN cofactor.