Apparent co-operativity for highly concentrated Michaelian and allosteric enzymes

Laurent, Michel; Kellershohn, Nicolas

doi:10.1016/0022-2836(84)90335-8

Cited by 7 publications

(6 citation statements)

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“…The magnitude of the apparent cooperativity increases as [ E ] T increases relative to K m , causing a concomitant increase in the substrate concentration ([ S ] T ) 0.5 at which half-maximal velocity is observed. 47 This increase is given by equation (2):

{false({false[S false]}_{T} false)}_{0.5} = \frac{{[E]}_{T}}{2} + K_{m}

…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, it has been shown that when the concentration of enzyme, [E] T , is equal to or greater than the K m , enzymes that follow Michaelis–Menten kinetics appear to have cooperative activity. The magnitude of the apparent cooperativity increases as [E] T increases relative to K m , causing a concomitant increase in the substrate concentration ([S] T ) 0.5 at which half-maximal velocity is observed . This increase is given by eq : false( false[ normalS false] normalT false) 0.5 = false[ normalE false] normalT 2 + K normalm Eq can be used to calculate K m from a plot of reaction velocity versus substrate concentration for enzymes operating under mutual depletion conditions. , …”

Section: Resultsmentioning

confidence: 99%

“…There are several plausible explanations for these deviations from standard hyperbolic Michaelis-Menten kinetics. First, the sigmoidal shape of the kinetic data may be an artifact of the high enzyme concentration used in these experiments, 47 as it was necessary for the concentrations of YoeB Sa 1 and the fluorogenic substrate to be roughly equal (5 μM and 0.5–20 μM, respectively) in order to observe YoeB Sa 1 activity using the fluorometric assay. Alternatively, a slow transition between inactive and active enzyme conformations may be required for catalysis, resulting in failure to achieve a steady-state of bound substrate due to the faster speed of enzymatic catalysis.…”

Section: Discussionmentioning

confidence: 99%

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Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Larson

Hergenrother

2013

Biochemistry

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The Staphylococcus aureus chromosome harbors two homologues of the YefM-YoeB toxin-antitoxin (TA) system. The toxins YoeBSa1 and YoeBSa2 possess ribosome-dependent ribonuclease (RNase) activity in Escherichia coli. This activity is similar to that of the E. coli toxin YoeBEc, an enzyme that, in addition to ribosome-dependent RNase activity, possesses ribosome-independent RNase activity in vitro. To investigate whether YoeBSa1 is also a ribosome-independent RNase, YoeBSa1 was expressed using a novel strategy, and its in vitro RNase activity, sequence specificity, and kinetics were characterized. Y88 of YoeBSa1 was critical for in vitro activity and cell culture toxicity. This residue was mutated to ortho-nitrobenzyl tyrosine (ONBY) via unnatural amino acid mutagenesis. YoeBSa1-Y88ONBY could be expressed in the absence of the antitoxin YefMSa1 in E. coli. Photocaged YoeBSa1-Y88ONBY displayed UV light-dependent RNase activity toward free mRNA in vitro. The in vitro ribosome-independent RNase activity of YoeBSa1-Y88ONBY, YoeBSa1-Y88F, and YoeBSa1-Y88TAG was significantly reduced or abolished. In contrast to YoeBEc, which cleaves RNA at both adenosine and guanosine with a preference for adenosine, YoeBSa1 cleaved mRNA specifically at guanosine. Using this information, a fluorometric assay was developed and used to determine the kinetic parameters for ribosome-independent RNA cleavage by YoeBSa1.

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{false({false[S false]}_{T} false)}_{0.5} = \frac{{[E]}_{T}}{2} + K_{m}

…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Larson

Hergenrother

2013

Biochemistry

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“…Attempts have been made to deal with situations of this type for enzyme catalysis (Halfman and Marcus, 1982;Laurent and Kellershohn, 1984;Kellershohn and Laurent, 1985). This concentration effect has been shown to modify profoundly the co-operative properties of multimeric enzymes.…”

Section: Introductionmentioning

confidence: 99%

Prion Diseases: Dynamics of the Infection and Properties of the Bistable Transition

Kellershohn

Laurent

2001

Biophysical Journal

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Prion diseases are thought to result from a pathogenic, conformational change in a cellular protein, the prion protein. The pathogenic isoform seems to convert the normal isoform in an autocatalytic process. In contrast to the conditions used for in vitro studies of enzyme kinetics, the concentration of the catalyst is not much lower than that of the substrate in the course of infection. This feature may endow the system with a time-hierarchy allowing the pathogenic isoform to relax very slowly in the course of infection. This may contribute to the long incubation periods observed in prion diseases. The dynamic process of prion propagation, including turnover of the cellular prion protein, displays bistable properties. Sporadic prion diseases may result from a change in one of the parameters associated with metabolism of the prion protein. The bistable transition observed in sporadic disease is reversible, whereas that observed in cases of exogenous contamination is irreversible. This model is consistent with the occurrence of rare, sporadic forms of prion diseases. It may also explain why only some individuals of a cohort develop a prion disease following transient food contamination.

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“…Thus there is much current interest in the study of highly concentrated enzymes. High-enzyme-concentration effects are particularly important for understanding the regulatory and amplification properties of enzymes (Laurent & Kellershohn, 1984). However, under these conditions, initial velocities can hardly be measured, since depletion of substrate cannot be neglected when enzyme is highly concentrated, i.e.…”

Section: Introductionmentioning

confidence: 99%

Analysis of progress curves for a highly concentrated Michaelian enzyme in the presence or absence of product inhibition

Kellershohn¹,

Laurent²

1985

Biochemical Journal

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Methods are given for analysing the time course of an enzyme-catalysed reaction when the concentration of the enzyme itself is high, a situation which is often found in vivo. (1) The integrated form of the kinetic equation for a concentrated Michaelian enzyme in absence of product inhibition is given. Parameters are shown to be calculated easily using non-linear fitting procedures. (2) A general algorithm to analyse progress-curve data in more complex cases (i.e. when the analytical form of the integrated rate equation is not known or is exceedingly complex) is proposed. This algorithm may be used for any enzyme mechanism for which the differential form of the kinetic equation may be written analytically. We show that the method allows differentiation between the main types of product inhibition which may occur in the case of a highly concentrated Michaelian enzyme.

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Apparent co-operativity for highly concentrated Michaelian and allosteric enzymes

Cited by 7 publications

References 22 publications

Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Prion Diseases: Dynamics of the Infection and Properties of the Bistable Transition

Analysis of progress curves for a highly concentrated Michaelian enzyme in the presence or absence of product inhibition

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Apparent co-operativity for highly concentrated Michaelian and allosteric enzymes

Cited by 7 publications

References 22 publications

Light Activation of Staphylococcus aureus Toxin YoeBSa1 Reveals Guanosine-Specific Endoribonuclease Activity

Light Activation of Staphylococcus aureus Toxin YoeBSa1 Reveals Guanosine-Specific Endoribonuclease Activity

Prion Diseases: Dynamics of the Infection and Properties of the Bistable Transition

Analysis of progress curves for a highly concentrated Michaelian enzyme in the presence or absence of product inhibition

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Product

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Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity