Transient Enzyme Kinetics at High Pressure

Author(s):  
Claude Balny

In a detailed study of an enzyme reaction pathway, a measured composite rate constant, for example, kcat, can be interpreted in ways that lead to ambiguous conclusions. Two conditions must be met to solve this problem: (1) an elementary rate constant must be measured, and (2) a maximum number of physical-chemical parameters must be used to perturb the system under study. To gain access to elementary rate constants, cryobaroenzymology and/or transient methods, such as stopped-flow and flow-quench kinetics, can be used. Both perturbation and kinetics measurements performed under either high pressure or low temperatures can then be used to probe the thermodynamics of the interconversion of two successive intermediates to obtain parameters such as ΔG‡, ΔS‡, ΔH‡, and ΔV‡ The interdependence of the two major variables, namely temperature and pressure, is presented in this article, in which the role of organic cosolvents is considered as a third variable. During catalytic reactions, enzymes undergo a number of conformational changes related to their dynamic structural flexibility. This appears as a succession of different steps. A complete study of such processes, which generally are very rapid, consists of the exploration of the properties of these steps, including thermodynamic features obtained by the action of temperature and pressure. As long ago as 1950, Laidler (1950) formulated the first theoretical basis for explaining the responses of enzymes to high hydrostatic pressures. Chemists used this parameter extensively, and in the early stages of high-pressure kinetics they attempted to analyze the observed results on the basis of collision theory (Asano, 1991) or transition-state theory (Evans & Polanyi, 1935). These theories are still used to describe pressure effects on enzyme reactions. It is postulated that between two successive intermediates there is a labile transition state which governs the energetics of the reaction (Glastone et al., 1941). But we must remember that this theory was first applied only to simple homogeneous reactions in gases. For solutions, the treatment can require the introduction of other parameters such as the viscosity.

1991 ◽  
Vol 69 (11) ◽  
pp. 1699-1704 ◽  
Author(s):  
P. T. T. Wong

Hydrogen/deuterium (H/D) exchange rate constants in chymotrypsinogen have been determined at several pressures up to 28.9 kbar by FTIR spectroscopy. The secondary structure of the protein molecules was monitored simultaneously at the corresponding pressures by the intensity redistribution of the infrared amide I band at these pressures. As in other proteins, the labile protons on the amide groups in chymotrypsinogen can, to a good approximation, be separated into two classes, each with distinct first order H/D exchange rates constants in the time period from 10 min to ~24 h. The fast exchange rate constant increases while the slow exchange rate constant decreases with increasing pressure. The increase in the fast exchange rate constant at high pressure is largely associated with the pressure-induced unfolding of the protein molecules. At extremely high pressure (12.8 kbar), in addition to the unfolding of protein molecules, pressure induced a distortion and weakening of the hydrogen bonds of the fold protein segments also contribute to an increase in the overall H/D exchange rate. The present results confirm that when chymotrypsinogen is dissolved in D2O, a considerable amount of D2O molecules is bound to the protein molecules on the surface as well as in the interior cavities of the molecules. The H/D exchange takes place between these bound D2O and the protons in the protein molecules. The mechanism of the H/D exchange and the interior dynamics in proteins are discussed on the basis of the present results. Key words: hydrogen/deuterium exchange, exchange kinetics, rate constant, pressure effects, infrared spectroscopy, protein, conformation structure, bound water.


2014 ◽  
Vol 43 (14) ◽  
pp. 5274-5279 ◽  
Author(s):  
O. Troeppner ◽  
D. Huang ◽  
R. H. Holm ◽  
I. Ivanović-Burmazović

The thermodynamics and high-pressure kinetics of the fastest CO2 fixation reaction by a metal-bound hydroxide resulted in a clear mechanistic picture and characterization of a very compact five-coordinate transition state.


2021 ◽  
Author(s):  
Chiwook Park

kcat and kcat/KM are the two fundamental kinetic parameters in enzyme kinetics. kcat is the first-order rate constant that determines the reaction rate when the enzyme is fully occupied at a saturating concentration of the substrate. kcat/KM is the second-order rate constant that determines the reaction rate when the enzyme is mostly free at a very low concentration of the substrate. Both parameters provide critical information on how the enzyme lowers the energy barriers along the reaction pathway for catalysis. However, it is surprising how often kcat/KM is used inappropriately as a composite parameter derived by dividing kcat with KM to assess both catalytic power and affinity to the substrate of the enzyme. The main challenge in explaining the true meaning of kcat/KM is the difficulty to demonstrate how the reaction energetics of enzyme catalysis determines kcat/KM in a simple way. Here, I report a step-by-step demonstration on how to visualize the meaning of kcat/KM on the reaction energy diagram. By using the reciprocal form of the expression of kcat/KM with the elementary rate constants in kinetic models, I show that kcat/KM is a harmonic sum of several kinetic terms that correspond to the heights of the transition states relative to the free enzyme. Then, I demonstrate that the height of the highest transition state has the dominant influence on kcat/KM, i. e. the step with the highest transition state is the limiting step for kcat/KM. The visualization of the meaning of kcat/KM on the reaction energy diagram offers an intuitive way to understand all the known properties of kcat/KM, including the Haldane relationship.


2010 ◽  
Vol 85 (7-9) ◽  
pp. 964-968 ◽  
Author(s):  
Xie Mao-lin ◽  
Luo De-li ◽  
Xian Xiao-bin ◽  
Leng Bang-yi ◽  
Chen Chang’an ◽  
...  

Author(s):  
Roland Winter ◽  
Anne Landwehr

Phospholipids, which provide valuable model systems for lipid membranes, display a variety of polymorphic phases, depending on their molecular structure and on environmental conditions. High hydrostatic pressure has been used as a physical parameter to study the thermodynamic properties and phase behavior of these systems. High pressure is also a characteristic feature of certain natural membrane environments. In the first part of this article, we review our recent work on the temperature- and pressure-dependent phase behavior of phospholipid systems differing in lipid conformation and headgroup structure. In the second part, we report on the determination of the (T, x, p) phase diagrams of binary phospholipid mixtures. An additional section deals with effects of incorporating ions, small amphiphilic molecules, and steroids into the bilayer on the experimental temperature- and pressure-dependent phase behavior of lipid systems. Finally, we discuss lamellar to nonlamellar thermotropic and barotropic phase transformations, which occur for a number of lipids, such as phosphatidylethanolamines, monoacylglycerides, and lipid mixtures. It has been suggested that nonlamellar lipid structures might play an important role as transient and local intermediates in a number of biochemical processes. High-pressure smallangle x-ray (SAXS) and neutron (SANS) scattering, differential scanning calorimetry (DSC), high-pressure differential thermal analysis (DTA), and p, V, T measurements have been used as experimental methods for the investigation of these systems. Lipid bilayer dispersions, in particular the phosphatidylcholines and phosphatidylethanolamines, are the workhorses for the investigation of biophysical properties of membrane lipids because they constitute the basic structural component of biological membranes. They exhibit a rich lyotropic and thermotropic phase behavior (Cevc & Marsh, 1987; Marsh, 1991; Yeagle, 1992). Most fully hydrated saturated phospholipid bilayers exhibit two principal thermotropic lamellar phase transitions, corresponding to a gel to gel (Lβ′–Pβ′) transition and a gel to liquid-crystalline (Pβ′–Lα) main transition at a temperature Tm. In the fluid-like La phase, the hydrocarbon chains of the lipid bilayers are conformationally disordered, whereas in the gel phases the hydrocarbon chains are more extended and relatively ordered.


Author(s):  
Dexter B. Northrop

Pressure effects on enzyme-catalyzed reactions were traditionally interpreted within a simplistic kinetic mechanism in which the transition state presented the highest energy barrier, and the chemical transformation of substrate to product was considered to be a singular, rate-limiting step. This was also true of isotope effects on enzyme-catalyzed reactions, but extensive isotopic studies have led to the conclusion that this transition state is rarely the highest barrier. Rather, the release of products (or the conformational change preceding product release) is usually the slowest step, often accompanied by several other partially ratelimiting steps. Thus, our interpretations of pressure effects must be shifted accordingly. Values attributed to AV‡ have been determined for more than 50 enzymes, more of them with a positive sign than negative, and most in the range of 20 to 40 ml mol–1 but these may or may not have anything to do with the activation volume associated with the transition state. Volume changes specifically associated with the binding of ligands to enzymes have been reported as well, including some very large values, as high as ΔV = 85 ml mo–1. Kinetically, these equilibrium pressure effects also originate in conformational changes because water is not very compressible; hence, rates of diffusion to and from enzymes are virtually unaffected by pressure. Much larger changes, as high as ΔV = –391 mi mo–1, have been observed during disaggregation and denaturation of enzymes. Thus, while it is possible for pressure effects to be expressed on every step of an enzymatic reaction, and to cause denaturation as well, making kinetic data from pressure effects hopelessly complex and uninterpretable, it appears likely that the most significant pressure effects will be expressed on conformational changes associated with product dissociations, without much kinetic complexity. This makes sense from another point of view—that the largest volume changes are probably on solvation equilibria during ligand binding and protein folding. Pressure effects on isotope effects have the potential of specifically identifying whether or not a volume change occurs upon attaining the transition state. With the exception of hydrogen tunneling, intrinsic isotope effects are independent of pressure.


Author(s):  
Roland Winter ◽  
C. Czeslik

Lipid systems, which provide valuable model systems for biological membranes, display a variety of polymorphic phases, depending on their molecular structure and environmental conditions. By use of X-ray and neutron diffraction the temperature- and pressure-dependent structure and phase behavior of lipid systems, differing in chain configuration and headgroup structure, have been studied. Besides lamellar phases also nonlamellar phases have been investigated. Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of lyotropic lipid mesophases, but also because high pressure is an important feature of certain natural membrane environments (e.g., marine biotopes) and because the high pressure phase behavior of biomolecules is of biotechnological interest (e.g., high pressure food processing). We demonstrate that temperature and pressure have noncongruent effects on the structural and phase behavior. By using the pressure-jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was also investigated. The time constants for completion of the transitions depend on the direction of the transition, the symmetry and topology of the structures involved, and also on the pressure-jump amplitude. In addition, the effect of incorporating ions, steroids and polypeptides into bilayers on the temperature- and pressure-dependent phase behavior of the lipid systems is discussed.


1977 ◽  
Vol 66 (2) ◽  
pp. 875-876 ◽  
Author(s):  
M. Lamotte ◽  
S. Risemberg ◽  
A. M. Merle ◽  
J. Joussot‐Dubien

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