Steady-state and pre-steady-state kinetics of the mitochondrial F(1)F(o) ATPase: is ATP synthase a reversible molecular machine?

H(+)-ATP synthase (F(1)F(o) ATPase) catalyzes the synthesis and/or hydrolysis of ATP, and the reactions are strongly affected by all the substrates (products) in a way clearly distinct from that expected of a simple reversibly operating enzyme. Recent studies have revealed the structure of F(1), which is ideally suited for the alternating binding change mechanism, with a rotating gamma-subunit as the energy-driven coupling device. According to this mechanism ATP, ADP, inorganic phosphate (P(i)) and Mg(2+) participate in the forward and reverse overall reactions exclusively as the substrates and products. However, both F(1) and F(1)F(o) demonstrate non-trivial steady-state and pre-steady-state kinetics as a function of variable substrate (product) concentrations. Several effectors cause unidirectional inhibition or activation of the enzyme. When considered separately, the unidirectional effects of ADP, P(i), Mg(2+) and energy supply on ATP synthesis or hydrolysis may possibly be explained by very complex kinetic schemes; taken together, the results suggest that different conformational states of the enzyme operate in the ATP hydrolase and ATP synthase reactions. A possible mechanism for an energy-dependent switch between the two states of F(1)F(o) ATPase is proposed.

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Effect of cations and anions on the steady state kinetics of energy-dependent Ca2+ transport in rat liver mitochondria.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)33154-x ◽

1976 ◽

Vol 251 (17) ◽

pp. 5251-5258

Author(s):

S M Hutson ◽

D R Pfeiffer ◽

H A Lardy

Keyword(s):

Steady State ◽

Rat Liver ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Steady State Kinetics ◽

Energy Dependent ◽

Kinetics Of

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Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-D-glucoside by cellobiase of Trichoderma viride

Canadian Journal of Biochemistry ◽

10.1139/o77-003 ◽

1977 ◽

Vol 55 (1) ◽

pp. 19-26 ◽

Cited By ~ 24

Author(s):

R. James Maguire

Keyword(s):

Steady State ◽

Trichoderma Viride ◽

Solution Temperature ◽

Temperature Range ◽

A Value ◽

Steady State Kinetics ◽

Decreasing Ph ◽

Ph Curve ◽

Kinetics Of ◽

Hydrolysis Of

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol–buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 × 10−3 M to 9 × 10−2 M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate – pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol−1 over the temperature range 5–56 °C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol.The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-β-D-glucoside was examined by pre-steady-state methods in which [Formula: see text], and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 s−1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism [Formula: see text]) (ii) a log rate–pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5–6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol−1 at pH 5.7 over the temperature range 10–60 °C.

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