The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

Nuiry, Ileana I.; Cook, Paul F.

doi:10.1016/0167-4838(85)90201-8

Cited by 8 publications

(5 citation statements)

References 8 publications

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“…The search for a model that meets the criteria for a holistic mechanistic model for SrtA catalysis led us to consider the possibility of reverse protonation. This type of mechanism has been invoked for an increasing number of enzymes, including fumarase, malic enzyme, thermolysin, β-xylosidase, and enolase which employ acid or base catalysis in the forward or reverse reaction (45,(59)(60)(61)(62). The underlying principle is that when two ionizable groups are required for catalysis and a bell-shaped pH-rate profile indicates that one residue must be protonated and the other deprotonated for activity, it is not possible to determine on the basis of the pH-rate profile alone what their respective protonation states are in the active form of the enzyme (60,62).…”

Section: Resultsmentioning

confidence: 99%

“…This type of mechanism has been invoked for an increasing number of enzymes, including fumarase, malic enzyme, thermolysin, β-xylosidase, and enolase which employ acid or base catalysis in the forward or reverse reaction (45,(59)(60)(61)(62). The underlying principle is that when two ionizable groups are required for catalysis and a bell-shaped pH-rate profile indicates that one residue must be protonated and the other deprotonated for activity, it is not possible to determine on the basis of the pH-rate profile alone what their respective protonation states are in the active form of the enzyme (60,62). Although at physiological pH the majority of the enzyme population will exist with the group with a lower pK a deprotonated and the group with a higher pK a protonated, there is an equilibrium in which a minor amount of enzyme exists in the reverse protonation state; i.e., the group with the lower pK a is protonated and the group with the higher pK a is deprotonated.…”

Section: Resultsmentioning

confidence: 99%

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Staphylococcus aureus Sortase Transpeptidase SrtA: Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism

et al. 2005

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The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Staphylococcus aureus Sortase Transpeptidase SrtA: Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism

et al. 2005

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“…For NAD-malic enzyme from Ascaris suum, the protonation state of the groups observed in the V/K profiles can be unambigiously assigned from the data presented. This has recently been accomplished for the chicken liver enzyme from a comparison of the pH dependence of the kinetic parameters in both reaction directions (Nuiry & Cook, 1985). In the present studies however, enough information is available in the direction of oxidative decarboxylation to make the assignment.…”

Section: Doublementioning

confidence: 91%

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide malic enzyme reaction from isotope effects and pH studies

Kiick¹,

Harris²,

Cook³

1986

Biochemistry

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The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.

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“…The favorable direction of the malate dehydrogenase (oxaloacetate decarboxylating) reaction is decarboxylation. However, some malic enzymes demonstrated a pH-dependence of this favorable reaction direction (to PA or malate formation) [ 146 ]. In addition to this, malic enzyme was shown to be a key enzyme in replenishing TCA cycle intermediates in Synechocystis sp.…”

Section: Part 2: Integration Of Tca Cycle Replenishing Pathways Separation Of Them On the Basis Of Main Metabolitesmentioning

confidence: 99%

“…As mentioned above, some carboxylyases (decarboxylases) direct the reaction depending on the environmental conditions (substrate/product concentrations, pH, etc.) [ 146 , 147 ]. The reversion of reaction 18 (or 18a) could lead to the formation of aconitate from acetate and PA.…”

Section: Part 2: Integration Of Tca Cycle Replenishing Pathways Separation Of Them On the Basis Of Main Metabolitesmentioning

confidence: 99%

TCA Cycle Replenishing Pathways in Photosynthetic Purple Non-Sulfur Bacteria Growing with Acetate

Petushkova

Mayorova

Tsygankov

2021

Life

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Purple non-sulfur bacteria (PNSB) are anoxygenic photosynthetic bacteria harnessing simple organic acids as electron donors. PNSB produce a-aminolevulinic acid, polyhydroxyalcanoates, bacteriochlorophylls a and b, ubiquinones, and other valuable compounds. They are highly promising producers of molecular hydrogen. PNSB can be cultivated in organic waste waters, such as wastes after fermentation. In most cases, wastes mainly contain acetic acid. Therefore, understanding the anaplerotic pathways in PNSB is crucial for their potential application as producers of biofuels. The present review addresses the recent data on presence and diversity of anaplerotic pathways in PNSB and describes different classifications of these pathways.

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The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

Cited by 8 publications

References 8 publications

Staphylococcus aureus Sortase Transpeptidase SrtA: Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism

Staphylococcus aureus Sortase Transpeptidase SrtA: Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide malic enzyme reaction from isotope effects and pH studies

TCA Cycle Replenishing Pathways in Photosynthetic Purple Non-Sulfur Bacteria Growing with Acetate

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