[29] Kinetic behavior of immobilized enzyme systems

Goldstein, Léon

doi:10.1016/s0076-6879(76)44031-4

Cited by 218 publications

(87 citation statements)

References 112 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Deviations from linearity for such plots are common with insoluble enzymes (Havekes et al, 1974;Campbell and Hornby, 1975;Engasser et al, 1977;Lluis, 1984) and may reflect a diffusion-limited step (Goldstein, 1976). Although the explanation of the downward curvature of the double-reciprocal plots with ubiquinol is not straightforward in a diffusion-limited model, it may be empirically compatible with diffusional limitation by cytochrome c. Analogous patterns are exhibited by immobilized enzymes (Havekes et al, 1974;Campbell and Hornby, 1975;Lluis, 1984), although a clear demonstration that the behaviour was actually a manifestation of diffusion control was not given in these studies.…”

Section: Saturation Kineticsmentioning

confidence: 99%

Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects

Fato

Cavazzoni

Castelluccio

et al. 1993

Biochemical Journal

View full text Add to dashboard Cite

In an attempt to establish the relative importance of diffusional and chemical control in the reactivity of the two substrates, ubiquinol and cytochrome c, we have undertaken an extensive characterization of the steady-state kinetics of ubiquinol-cytochrome c reductase (EC 1.10.2.2) when present in open submitochondrial particles from bovine heart. The kinetic pattern follows a Ping Pong mechanism; contrary to the situation found with the isolated enzyme [Speck and Margoliash (1984) J. Biol. Chem. 259, 1064-1072, and confirmed in our laboratory], no substrate inhibition by oxidized cytochrome c was observed with the membrane-bound enzyme. Endogenous oxidized ubiquinone-10 is unable to exert product inhibition under the conditions employed. In the Ping Pong mechanism for this enzyme, the reaction scheme can be clearly divided into two parts, and the Kmin (keat/Km) value for one substrate is independent of the rate constant for the second substrate. Both ubiquinol-1 and ubiquinol-2 can be used as electron donors reacting with the enzyme from within the lipid bilayer [Fato, Castelluccio, Palmer and Lenaz (1988) Biochim. Biophys. Acta 932,[216][217][218][219][220][221][222]; the kmin values for ubiquinols, when calculated on the basis of their membranous concentrations, are significantly lower than the kmin for cytochrome c. The temperature-dependence of the kinetic parameters was investigated by titrating each of the substrates under quasi-saturating concentrations of the second substrate. Arrhenius plots of Vm.. extrapolated from both cytochrome c and ubiquinol titrations were linear, when care was taken to verify the quasi-saturating concentrations of the fixed co-substrate. The Arrhenius plots for the kmin values for both ubiquinol and cytochrome c were linear, but the activation energy was much higher for the former, particularly when calculated for ubiquinol dissolved in the lipid phase; the very low value of activation energy of the kmin for cytochrome c is strong support for diffusion control being present in the reaction of cytochrome c with the membranous enzyme. In contrast to the soluble enzyme, ubiquinone titrations of submitochondrial particles at low cytochrome c concentrations deviated from hyperbolic behaviour. Changing the medium viscosity with either poly(ethylene glycol) or sucrose had a strong effect on the cytochrome c kmin, whereas the change in the ubiquinol kmin was much smaller. From the viscosity studies the extent of diffusional control could be calculated, revealing that the reaction with cytochrome c was mostly diffusion-limited. The viscosity of the membrane was changed by incorporating cholesterol; no significant effect on the ubiquinol kmin ascribable to diffusion control could be recognized. The bulk of the studies reported here strongly suggest that ubiquinol-cytochrome c reductase is subject to diffusion control of the interaction of cytochrome c from the aqueous medium, but not of the interaction of the short-chain ubiquinols from the membrane interior. INTRODUCTIONSeveral enzy...

show abstract

Section: Saturation Kineticsmentioning

confidence: 99%

Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects

Fato

Cavazzoni

Castelluccio

et al. 1993

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Surface immobilization, which can extend the observation time, may introduce disturbances to the function of the molecules (12,13) or even may require cross-linking of multisubunit complexes to prevent surface-induced degradation (14). To address this problem, a device termed the Anti-Brownian ELectrokinetic trap (ABEL trap) has been developed as a noncontacting approach for extending the observation time of single fluorescent biomolecules in free solution (15).…”

mentioning

confidence: 99%

Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution

Jiang

Douglas²,

Conley³

et al. 2011

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

View full text Add to dashboard Cite

In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperoninbound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.single molecule | allostery | fluorescence | enzymology | nucleotide counting Y ears of study of cooperativity for proteins with multiple ligand binding sites have yielded important insights, ever since the role of cooperative oxygen binding to hemoglobin in high oxygendelivery throughput was recognized (1-3). Multisubunit enzymes usually hydrolyze ATP in a concerted fashion, but actually observing this process, enzyme by enzyme, can provide a deeper picture of the underlying cooperativity resulting from communication among the various subunits. A well known example has been the study of the rotary motor F1-ATPase by single-molecule techniques (4). Another class of multisubunit cellular machines is the double-ring type I and type II chaperonins which can have from 14 to 18 subunits each of which can hydrolyze ATP (5, 6). Ensemble studies of the bacterial type I GroEL/GroES system, for example, have indicated that ATP binds subunits in one 7-membered ring with positive cooperativity, while negative cooperativity operates between rings (7). We focus on the cooperative process operative in the mammalian type II chaperonin TRiC/CCT, which has two ring-shaped cavities with built-in lids composed of eight different subunits each. TRiC is essential for the folding of a number of key proteins in mammalian cells, including actin, tubulin, and many cell cycle regulators (8). Previous ensemble measurements of ATP-induced allosteric transition rates and steady-state ATPase rate in TRiC show evidence for positive and negative cooperativity du...

show abstract

“…Amongst possible reasons are mass transfer and diffusion limitations of the substrate, product or effectors molecules between the bulk phase and the surface, together with changes in protein conformation resulting in altered physical properties of the active site (Engasser and Horvath, 1973;1974a;1974b;1974c;Goldstein, 1976;Taylor and Swaisgood, 1981).…”

Section: Disscusionmentioning

confidence: 99%

Tailor-Made Enzyme Carriers: Preparation and Use of Adsorbents Specifically Designed to Immobilize Allosteric Enzymes in Activated Conformation

Salemi¹

2010

American Journal of Biochemistry and Biotechnology

View full text Add to dashboard Cite

Problem statement:The enzyme immobilization has experienced substantial growth in the recent past and an ever increasing amount of study has been reported on various aspects of immobilized enzymes. In most of these investigations, catalytic activities are found to be diminished as compared to the enzyme free in solution. Approach: Hydrophobic adsorbents were prepared containing L-leucine or citric acid, two positive allosteric effectors, for bovine liver Glutamate Dehydrogenase (GDH, EC 1.4.1.3) and heart mitochondrial Malate Dehydrogenase (MDH, EC 1.1.1.37 ), respectively. Results: Immobilized preparations of these well-defined allosteric enzymes indicated improved catalytic activities as compared with those involving use of the adsorbents without these activators. Conclusion/Recommendations: It is concluded that the regulatory proteins are Furthermore; they retain their natural capacity for undergoing the conformational transitions needed for enhanced catalytic activities. Adsorptive immobilization of these two allosteric proteins in activated conformation may serve as useful models in relation to design strategies for preparation of tailor-made enzyme carriers.

show abstract

[29] Kinetic behavior of immobilized enzyme systems

Cited by 218 publications

References 112 publications

Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects

Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects

Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution

Tailor-Made Enzyme Carriers: Preparation and Use of Adsorbents Specifically Designed to Immobilize Allosteric Enzymes in Activated Conformation

Contact Info

Product

Resources

About