Sunil Nath

A SYSTEMATIC APPROACH FOR INVESTIGATION OF SPRAY DRYING PROCESSES

Abstract A systematic approach is developed for investigation of spray drying processes for chemical and biological systems. The approach involves an in-depth study of atomizer performance, spray dryer parametric sensitivity, spray-dried powder properties, thermal inactivation and post-drying properties. The approach helps considerably in rational design of spray drying experiments and in investigation and optimization of various process aspects of spray drying of chemical and biological systems, leading to large savings in labor, cost and time.

Role of water structure on phase separation in polyelectrolyte-polyethyleneglycol based aqueous two phase systems

Abstract Partitioning of proteins in aqueous two-phase systems (ATPS) has emerged as one of the important downstream processing techniques in bioprocess technology. The phase separation behavior of polyelectrolyte–polyethyleneglycol (PEG) based ATPS have been studied to elucidate the mechanism controlling phase behavior. The effect of various inorganic salt additives revealed the importance of water structure as a major factor controlling phase separation in these systems. Nitrate and potassium (water structure breaking ions) elevated the binodial line while sulphate, phosphate and sodium (water structure making ions) depressed the binodial line in both polyacrylic acid–PEG as well as polyethylenimine–PEG based ATPS. The effect of increase in concentration of either of the constituent polymers in both systems (at constant salt concentration) always led to a greater propensity towards phase separation. These results point to a mechanism in which salt - assisted polymer - modified water structure interactions play a central role in phase separation in ATPS.

The Molecular Mechanism of ATP Synthesis by F1F0-ATP Synthase: A Scrutiny of the Major Possibilities

A critical goal of metabolism in living cells is the synthesis of adenosine triphosphate (ATP). ATP is synthesized by the enzyme F1F0-ATP synthase. This enzyme, the smallest-known molecular machine, couples proton translocation through its membrane-embedded, hydrophobic domain, F0, to the synthesis of ATP from adenosine diphosphate (ADP) and inorganic phosphate (Pi) in its soluble, hydrophilic headpiece, F1. Animals, plants and microorganisms all capture and utilize energy by this important chemical reaction. How does it occur? The binding change mechanism and the torsional mechanism of energy transduction and ATP synthesis are two mechanisms that have been proposed in the literature. According to the binding change mechanism (which considers reversible catalysis and site-site cooperativity), energy is required primarily for release of synthesized ATP, but not for its synthesis. On the other hand, according to the torsional mechanism (which considers an irreversible mode of catalysis and absence of cooperativity), all the elementary steps require energy, and the ion-protein interaction energy obtained from the ion gradients is used to synthesize ATP, for Pi binding, and for straining the beta-epsilon bond in order to enable ADP to bind. The energy to release preformed ATP from the tight catalytic site (betaDP) is provided by the formation of the beta-epsilon ester linkage. First, the central features of these mechanisms are clearly delineated. Then, a critical scrutiny of these mechanisms is undertaken. The predictions of the torsional mechanism are listed. In particular, how the torsional mechanism deals with the specific difficulties associated with other mechanisms, and how it seeks to explain a wealth of structural, spectroscopic, and biochemical data is discussed in detail. Recent experimental data in support of the mechanism are presented. Finally, in view of the molecular machine nature of energy transduction, the indispensability of applying engineering tools at the molecular level is highlighted. This paves the way for the development of a new field: Molecular Physiological Engineering.

A THERMODYNAMIC PRINCIPLE FOR THE COUPLED BIOENERGETIC PROCESSES OF ATP SYNTHESIS

Based on a nonequilibrium thermodynamic description of the coupled bioenergetic processes of ATP synthesis, I show for the first time that the entropy production depends on the physical system of coupling; completely delocalized coupling leads to minimum rate of entropy production in the steady state. Further, when nonlinear processes are considered, the free energy dissipation exhibits a minimum at the operating distance from equilibrium. These results agree with experimental observations. The principle proposed here accurately predicts the coupling system and the distance from equilibrium in fundamental life processes.

Kinetic model of ATP synthase: pH dependence of the rate of ATP synthesis

Recently, a novel molecular mechanism of torque generation in the F0 portion of ATP synthase was proposed [Rohatgi, Saha and Nath (1998) Curr. Sci. 75, 716–718]. In this mechanism, rotation of the c‐subunit was conceived to take place in 12 discrete steps of 30° each due to the binding and unbinding of protons to/from the leading and trailing Asp‐61 residues of the c‐subunit, respectively. Based on this molecular mechanism, a kinetic scheme has been developed in this work. The scheme considers proton transport driven by a concentration gradient of protons across the proton half‐channels, and the rotation of the c‐subunit by changes in the electrical potential only. This kinetic scheme has been analyzed mathematically and an expression has been obtained to explain the pH dependence of the rate of ATP synthesis by ATP synthase under steady state operating conditions. For a single set of three enzymological kinetic parameters, this expression predicts the rates of ATP synthesis which agree well with the experimental data over a wide range of pHin and pHout. A logical consequence of our analysis is that ΔpH and Δψ are kinetically inequivalent driving forces for ATP synthesis.

A rapid method for determining kinetic parameters of enzymes exhibiting nonlinear thermal inactivation bahavior

A rapid method is developed to analyze the kinetics of thermal inactivation of enzymes that exhibit a nonlinear biphasic log(activity)-time relationship. Thermal destruction experiments on alcohol dehydrogenase from bakers yeast demonstrate the applicability of the method. The method is based on physical considerations (as opposed to mathematical curve fitting/regression methods) and also serves as a quick check of results obtained using nonlinear regression. It is superior to fitting nonlinear enzyme inactivation data by first-order kinetics or taking the initial and final slopes of the inactivation data. In fact, the method is of general validity and can be applied to any decay process that can be represented by a sum of exponentials.

Beyond the Chemiosmotic Theory: Analysis of Key Fundamental Aspects of Energy Coupling in Oxidative Phosphorylation in the Light of a Torsional Mechanism of Energy Transduction and ATP Synthesis—Invited Review Part 1

The core of this second article shows how logical errors and inconsistencies in previous theories of energy coupling in oxidative phosphorylation are overcome by use of a torsional mechanism and the unified theory of ATP synthesis/hydrolysis. The torsional mechanism is shown to satisfy the pioneering and verified features of previous mechanisms. A considerable amount of data is identified that is incompatible with older theories but is now explained in a logically consistent and unified way. Key deficiencies in older theories are pinpointed and their resolution elucidated. Finally, major differences between old and new approaches are tabulated. The new theory now provides the elusive details of energy coupling and transduction, and allows several novel and experimentally verifiable predictions to be made and a considerable number of applications in nanotechnology, energy conversion, systems biology, and in health and disease are foreseen.

Oxidative phosphorylation revisited

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchells chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground‐breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Naths torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchells chemiosmotic theory pointed out by other authors. It is concluded that the energy‐transducing complexes in oxidative phosphorylation and photosynthesis are proton‐dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of oxidative phosphorylation and photophosphorylation from a wholly new perspective will rekindle scientific discussion of a key process in bioenergetics and catalyze new avenues of research in a truly interdisciplinary field. Biotechnol. Bioeng. 2015;112: 429–437.

Molecular Mechanisms of Energy Transduction in Cells: Engineering Applications and Biological Implications

The synthesis of ATP from ADP and inorganic phosphate by F1F0-ATP synthase, the universal enzyme in biological energy conversion, using the energy of a transmembrane gradient of ions, and the use of ATP by the myosin-actin system to cause muscular contraction are among the most fundamental processes in biology. Both the ATP synthase and the myosin-actin may be looked upon as molecular machines. A detailed analysis of the molecular mechanisms of energy transduction by these molecular machines has been carried out in order to understand the means by which living cells produce and consume energy. These mechanisms have been compared with each other and their biological implications have been discussed. The thermodynamics of energy coupling in the oxidative phosphorylation process has been developed and the consistency of the mechanisms with the thermodynamics has been explored. Novel engineering applications that can result have been discussed in detail and several directions for future work have been pointed out.

Catalysis by ATP synthase: mechanistic, kinetic and thermodynamic characteristics

Abstract Mechanistic, kinetic and thermodynamic aspects of ATP catalysis by ATP synthase have been determined and analyzed. Reversibility and irreversibility of catalysis in ATP synthase represent two contrasting modes of catalysis with important implications for the molecular mechanism of ATP synthesis. To shed light on these aspects, we have developed kinetic schemes for ATP synthesis and hydrolysis; analysis of these schemes reveals several novel features and provides new directions for further research. First, the ratio of bound 32 P i to total bound 32 P can be expressed in terms of the rate constants of the elementary catalytic steps, which are characteristic properties of the system; therefore, results of classical cold chase/acid quench 32 P i experiments interpreted in terms of an equilibrium distribution of bound substrate and product at the catalytic site can be explained by an irreversible mode of catalysis. Second, characterization of the mechanistic and kinetic properties reveals the absence of cooperativity in ATP synthase, and that product release precedes substrate binding. Third, ΔpH and Δ ψ are kinetically inequivalent in driving ATP synthesis, and Δ μ H is not the true driving force for ATP synthesis. Thermodynamic analysis of ATP synthesis reveals a dynamically electrogenic but overall electroneutral mode of ion transport across the membrane. The P/O ratio based on the torsional mechanism was obtained and was shown to explain the experimental observations of the past 50 years and to be in agreement with the thermodynamic calculations. Taken together, these findings necessitate a paradigm shift for understanding the molecular mechanism of ATP synthesis.