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Kuzmic P.,BioKin Ltd
Biochimica et Biophysica Acta - Proteins and Proteomics

A generalized numerical treatment of steady-state enzyme kinetics is presented. This new approach relies on automatic computer derivation of the underlying mathematical model (a system of simultaneous nonlinear algebraic equations) from a symbolic representation of the reaction mechanism (a system of biochemical equations) provided by the researcher. The method allows experimental biochemists to analyze initial-rate enzyme kinetic data, under the steady-state approximation, without having to use any mathematical equations. An illustrative example is based on the inhibition kinetics of p56lck kinase by an ATP competitive inhibitor. A computer implementation of the new method, in the modified software package DYNAFIT [Kuzmič, P. (1996) Anal. Biochem. 237, 260-273], is freely available to all academic researchers. © 2009. Source

Cle C.,John Innes Center | Martin C.,John Innes Center | Field R.A.,John Innes Center | Kuzmic P.,BioKin Ltd | Bornemann S.,John Innes Center
Biocatalysis and Biotransformation

Strategically important cellular components, such as the cell wall and the starch granule, present surfaces during their biosynthesis and degradation. The enzymology of such surfaces is experimentally challenging and goes well beyond classical solution-state analyses. The kinetics of surface catalysis is complex but tractable. A number of approaches to monitor surface catalysis are reviewed and each is suited to a different biological problem. Particular attention is paid to a method we have recently developed for quantitatively monitoring polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy. This method has many attractive features with the potential to tackle both biological and industrial problems. © 2010 Informa UK Ltd. Source

Optimal experimental designs for the dose-response screening of enzyme inhibitors were studied within the framework of the Box-Lucas theory. If the enzyme concentration E is considered as a fixed constant, an exact two-point D-optimal design consists of a pair of inhibitor concentrations equal to I1=0 and I2=E+K, where K is the apparent inhibition constant. If the enzyme concentration is treated as an adjustable parameter, an empirical three-point D-optimal design consists of three inhibitor concentrations equal to I1=0, I2=E+3K, and I3=0.7E. These results were applied to design optimized, irregularly spaced concentration series for routine inhibitor screening. A heuristic Monte Carlo simulation study confirmed that the optimized dilution series is significantly more efficient than the classic series characterized by a constant dilution ratio. An online calculator to create optimized dilution series is freely available at http://www.biokin.com/design/. © 2011 Elsevier Inc. All rights reserved. Source

Salykin A.,Masaryk University | Salykin A.,St Annes University Hospital Brno | Kuzmic P.,BioKin Ltd | Kyrylenko O.,Masaryk University | And 6 more authors.
Stem Cell Reviews and Reports

Recent evidence suggests that energy metabolism contributes to molecular mechanisms controlling stem cell identity. For example, human embryonic stem cells (hESCs) receive their metabolic energy mostly via glycolysis rather than mitochondrial oxidative phosphorylation. This suggests a connection of metabolic homeostasis to stemness. Nicotinamide adenine dinucleotide (NAD) is an important cellular redox carrier and a cofactor for various metabolic pathways, including glycolysis. Therefore, accurate determination of NAD cellular levels and dynamics is of growing importance for understanding the physiology of stem cells. Conventional analytic methods for the determination of metabolite levels rely on linear calibration curves. However, in actual practice many two-enzyme cycling assays, such as the assay systems used in this work, display prominently nonlinear behavior. Here we present a diaphorase/ lactate dehydrogenase NAD cycling assay optimized for hESCs, together with a mechanism-based, nonlinear regression models for the determination of NAD+, NADH, and total NAD. We also present experimental data on metabolic homeostasis of hESC under various physiological conditions. We show that NAD+/NADH ratio varies considerably with time in culture after routine change of medium, while the total NAD content undergoes relatively minor changes. In addition, we show that the NAD+/NADH ratio, as well as the total NAD levels, vary between stem cells and their differentiated counterparts. Importantly, the NAD+/NADH ratio was found to be substantially higher in hESC-derived fibroblasts versus hESCs. Overall, our nonlinear mathematical model is applicable to other enzymatic amplification systems. © 2013 Springer Science+Business Media New York. Source

Kleckner I.R.,Ohio State University | McElroy C.A.,Ohio State University | Kuzmic P.,BioKin Ltd | Gollnick P.,State University of New York at Buffalo | Foster M.P.,Ohio State University

The trp RNA-binding attenuation protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of trp-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both microsecond to millisecond rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of 11 bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by Bacillus stearothermophilus TRAP over a range of temperatures and observed well-separated kinetic steps. These data were analyzed using nonlinear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two-binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that dissociation of Trp from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature-dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery. © 2013 American Chemical Society. Source

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