Maximum catalytic activity of some key enzymes in provision of physiologically useful information about metabolic fluxes

Abstract

The energy required by tissues (e.g., for movement, biosynthesis, ion transport, and for maintenance of structures) is obtained at a molecular level by the hydrolysis of ATP to ADP. The resynthesis of ATP occurs in the processes of fuel oxidation within the cell. Since ATP is not stored in the cell, any increase in the rate of energy demand by a cell must result in an immediate and precise increase in the rate of fuel oxidation to provide the necessary ATP. In addition to these rapid regulatory responses, many animals and plants are subject to longer term changes in both the capacity for energy production and its utilisation by virtue of seasonal changes in environmental temperature, food availability, reproductive requirements, and behavioural demands. The modifications in metabolic pathways of energy storage, production, and utilisation with the progression of the seasons clearly enables organisms to take advantage of favourable conditions, as well as to survive particularly unfavourable conditions.Various fuels (e.g., glucose, glycogen, fatty acids, triglyceride, and amino acids) are available for oxidation by the individual tissues and these follow different routes or pathways of metabolism. One very important question for biologists and physiologists is: Which fuel(s) is being used to provide most of the energy for a given tissue? Indeed, knowledge of the maximum capacity of energy-producing pathways is required before the physiological importance of the pathway can be appreciated and, in some cases, before the physiological role of the tissue can be properly assessed.Various experimental techniques for the precise measurement of flux through a pathway have been developed largely over the past 30 years. A description of methods has been given elsewhere (Newsholme et al., '78; Newsholme et al., '80), but one of these, which depends on the measurement of the maximum activities of certain key enzymes, is described in detail in this article. This method has several advantages over others; it is very simple, it can be used for comparative studies of a large range of animals, and it can be used in humans (or any large animal), usually with minimum surgical intervention. Unfortunately, its very simplicit has led to its misapplication leading to a lac$ of confidence in the technique. For this reason, the theoretical practical basis for the choice of enzymes that can be used as flux indicators is described below, together with details of the practical approach to obtaining the quantitative information and, finally, some examples of its use. THEORETICAL BASIS FOR THE USE OF MAXIMUM ENZYME ACTIVITIES AS INDICATORS OF MAXIMUM FLUXBefore the reasons behind the choice of enzyme can be understood, the difference between near-equilibrium and nonequilibrium reactions must be explained, and the meaning of the term flux-generating step must be appreciated (for detailed reviews, see Newsholme and Crabtree, 1976,1979. Near-equilibrium, nonequilibrium and flux-generating reactionsNear-equilibrium and nonequilibrium reactionsReac...

Regulatory changes contribute to the adaptive enhancement of thermogenic capacity in high-altitude deer mice

“…For enzymes with large control coefficients, in vitro enzyme activity (V max ) can be used to estimate pathway flux capacity in vivo (45). Consistent with the gene expression patterns, activities for key enzymes in fatty-acid oxidation (β-hydroxyacyl-CoA dehydrogenase; HOAD) and OXPHOS (cytochrome c oxidase, COX) were significantly higher in highland deer mice compared with lowland deer mice and P. leucopus (Table 1).…”

Lifetime performance in foraging honeybees: behaviour and physiology

“…Of these, only CS showed increases in activity as bees gained foraging experience. Enzyme activities, or, more specifically, maximal enzymatic flux capacities (V max ), represent the upper limits of flux at a particular step of a biochemical pathway in vitro, but can provide insights on flux in vivo (Newsholme and Crabtree, 1986;Suarez et al, 1996;Suarez et al, 1997). V max is a function of enzyme concentration and catalytic efficiency (k cat ).…”

Relationships between enzymatic flux capacities and metabolic flux rates: Nonequilibrium reactions in muscle glycolysis

“…Enzymatic flux capacities in tissues have been defined in the literature as either V max ͞k m (e.g., refs. 31 and 32) or as V max (33,34). V max values obtained in vitro can be expressed in the same units as flux rates in vivo, making direct comparisons between these parameters possible.…”

“…Thus, in assessing capacity͞load relationships at the biochemical level, flux capacities are best represented by V max values (equal to [E] ϫ k cat , where [E] and k cat denote enzyme concentration and catalytic efficiency or turnover number, respectively) at each of the individual steps in pathways. The practice of measuring V max values under optimal conditions in vitro and relating these to in vivo flux rates is based on a sound theoretical framework (33,34). In combination with other information (e.g., intracellular substrate and product concentrations), V max values and physiological flux rates provide insights into enzyme function and regulation (e.g., refs.…”

“…16 and 35) in vivo. V max values serve as measures of upper limits to flux at specific steps in biochemical pathways (33,34). As such, they can be used as measures of functional capacities in analyses of biochemical design (36).…”