Metabolite Transfer via Enzyme-Enzyme Complexes

Srivastava, D. K.; Bernhard, Sidney A.

doi:10.1126/science.3775377

Cited by 271 publications

(124 citation statements)

References 28 publications

(40 reference statements)

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“…Assuming an average mammalian cellular protein content of ~0.75 ng (76), and an average volume of ~4 x 10 -12 liter (1), there would be ,~109 actin molecules/cell (72). This results in a cellular actin concentration of "d9 mg/ml, consistent with a total cellular protein concentration of '~ 200 mg/ml (66,67). If we assume that approximately half the total cellular volume is cytoplasmic, due to the volume fraction of mitochondria, vesicles, and endoplasmic reticulum, then the net actin concentration would be ,,040 mg/ml, or 900 tiM.…”

Section: How Much Aldolase Is In Cells?mentioning

confidence: 70%

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Pagliaro

Taylor

1988

The Journal of Cell Biology

115

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Abstract. We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 × 10 -7 cm2/s, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 × 10 -7 cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 × 10 -g cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITCdextran indicated that FITC-dextran was relatively excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.TIaOUGH glycolytic metabolism is well-defined at the biochemical level, the dynamic and spatial organization of glycolysis in living cells has not been characterized to date. After the elucidation of mitochondrial structure and function 35 years ago (28) it was generally assumed that a corresponding organelle for glycolysis must exist. Efforts to isolate and to characterize such an organelle were unsuccessful, leading de Duve (26) to conclude that the longsought"glycolytic particle" did not exist. As a result, the current concept of in vivo glycolytic metabolism is based on an assumption of dilute solution chemistry, due largely to the absence of evidence for a discrete glycolytic organelle.There has been evidence for over 20 years that some glycoiytic enzymes bind to structural proteins, particularly actin.

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Section: How Much Aldolase Is In Cells?mentioning

confidence: 70%

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Pagliaro

Taylor

1988

The Journal of Cell Biology

115

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“…These approaches are thus prone to two types of errors. First, they underestimate the functional consequences of modulating gene expression, especially for the many enzymes (19,47) whose normal activities have little influence on the steady-state flux of the embedding pathways. ‡ ‡ Second, they incorrectly predict that similar functional consequences ensue when changes in any two enzymes of the same metabolic branch yield the same change in flux.…”

Section: Discussionmentioning

confidence: 99%

“…Analytical calculations for a simple unbranched pathway (A.S. and M.A.S., unpublished data) and numerical results for a simple model pathway that was parameterized to resemble glycolysis (49) indicate that up-regulation of the equilibrium and downstream nonequilibrium enzymes achieves increased flux without the previous disadvantages. In metabolic engineering, it has also been found that large increases in some enzyme activities intended to ‡ ‡ If, as seems likely, the glycolytic pathway represents the typical situation, most enzymes are excess-activity enzymes, and these are usually also the most abundantly expressed enzymes in each pathway (19,47). Much, if not most, of the cellular protein with enzymatic activity may thus be fulfilling functional requirements other than flux capacity.…”

Section: Discussionmentioning

confidence: 99%

Evolution of enzymes in a series is driven by dissimilar functional demands

Salvador

Savageau

2006

Proc. Natl. Acad. Sci. U.S.A.

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That distinct enzyme activities in an unbranched metabolic pathway are evolutionarily tuned to a single functional requirement is a pervasive assumption. Here we test this assumption by examining the activities of two consecutively acting enzymes in human erythrocytes with an approach to quantitative evolutionary design that avoids the above-mentioned assumption. We previously found that avoidance of NADPH depletion during the pulses of oxidative load to which erythrocytes are normally exposed is the main functional requirement mediating selection for high glucose-6-phosphate dehydrogenase activity. In the present study, we find that, in contrast, the maintenance of oxidized glutathione at low concentrations is the main functional requirement mediating selection for high glutathione reductase activity. The results in this case show that, contrary to the assumption of a single functional requirement, natural selection for the normal activities of the distinct enzymes in the pathway is mediated by different requirements. On the other hand, the results agree with the more general principles that underlie our approach. Namely, that (i) the values of biochemical parameters evolve so as to fulfill the various performance requirements that are relevant to achieve high fitness, and (ii) these performance requirements can be inferred from quantitative systems theory considerations, informed by knowledge of specific aspects of the biochemistry, physiology, genetics, and ecology of the organism.human erythrocytes ͉ oxidative stress ͉ safety factors ͉ quantitative evolutionary design ͉ systems biology A lthough numerous mutations can drastically change the values of biochemical parameters such as enzyme activities, these values are often narrowly distributed in natural populations. This outcome owes largely to the interplay between constraints imposed by physicochemical laws and natural selection for good performance of the organism's biochemical circuits. The strength of natural selection in shaping the design of living organisms at the molecular level (1) is just beginning to be appreciated. In humans, natural selection is strong enough to almost prevent fixation of mutations that decrease fitness by Ͼ0.002%. Furthermore, Ͼ70% of the amino acid-changing mutations are selected against (2, 3). In the many organisms that have larger effective populations, even smaller fitness differentials can drive natural selection. Understanding the quantitative design for biochemical parameters that has been achieved through natural selection (i.e., the qualitative and quantitative functional requirements that mediate their evolutionary tuning) is a goal of systems biology with far-reaching implications. Such understanding would provide valuable insight regarding physiological as well as evolutionary adaptation (4, 5) and guidance for metabolic engineering (6). However, this type of understanding for parameters such as enzyme activities remains limited.The assumption that the distinct enzyme activities in a metabolic pathway are evolutio...

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“…Moreover in this expression U, and U , have the same value as in expression (20). The identity of U, in these two expressions implies that the ideal free energy of the dissociation of the complex into its constitutive enzymes be the same whether the enzyme El has bound the transition state S' or the transition state Xf.…”

Section: Thermodynamic Significance Of Generalized Microscopic Reversmentioning

confidence: 93%

“…Eqn (20) above would be equally valid if the association of the two enzymes were destabilizing the ground and (or) the transition states.…”

Section: Thermodynamic Significance Of Generalized Microscopic Reversmentioning

confidence: 99%

Coordination of catalytic activities within enzyme complexes

Kellershohn

Ricard

1994

European Journal of Biochemistry

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If two enzymes are physically and permanently associated as a bi-enzyme complex and if these enzymes catalyze non-consecutive chemical reactions, either of these reactions may inhibit or activate the other. If these reactions belong to two different metabolic cycles, the functioning of one of these cycles will control the fine tuning of the other.Thus simple kinetic considerations lead to the conclusion that, owing to the spatial organization of enzymes as multimolecular complexes, a fine tuning and a coordination of different metabolic networks, or cycles, may be exerted. It thus appears that channelling of reaction intermediates within a multienzyme complex does not represent the only functional advantage brought about by this type of spatial molecular organization.The view that is emerging from the quantitative study of the overall metabolic processes is that of a sort of 'molecular democracy' [l], that is, the various enzyme reactions of the same metabolic network cooperate as to maintain the fine tuning of that process [1-101. It is however evident that these metabolic networks, or cycles, have to be regulated one with respect to the other. There must therefore exist a coordination between these different metabolic processes. This coordination is usually believed to be exerted thanks to metabolic intermediates, such as ATP, NAD, NADP, etc., which appear in many metabolic networks and cycles. One may wonder, however, whether the presence of these intermediates common to different metabolic processes is sufficient p e r se to maintain a strict coordination between these metabolic processes.The existence of enzyme compartmentalization and spatial organization in the living cell offers a new possibility of understanding how this coordination may be effected. It is often considered that the functional advantage brought about by enzyme association is the existence of channelling of the reaction intermediates between the different catalytic sites within the multienzyme complex [11-281. This view is often substantiated by the experimental finding that the enzymes that are associated as a complex catalyze consecutive reactions [13-211. The reality of channelling is often difficult to prove experimentally [20-251. Moreover, in a number of cases, enzymes that do not catalyze consecutive reactions exist under the form of multimolecular complexes. This is the case, for instance, of a complex made up of glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase of chloroplasts [29, 301; of fructose 1,6-bisphosphatase and phosphoribulokinase isolated from a photosynthetic bacterium [31], of up to nine aminoacyl-tRNA synthetases in higher eukaryotes [32-371, of isoleucyl-tRNA synthetase and threonine deaminase [38] and of ornithine carbamoyltransferase and arginase [39]. Therefore if there is a funcCorrespondence to J. Ricard,

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Metabolite Transfer via Enzyme-Enzyme Complexes

Cited by 271 publications

References 28 publications

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Evolution of enzymes in a series is driven by dissimilar functional demands

Coordination of catalytic activities within enzyme complexes

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