A slow conformation change in the transient state kinetics of soluble ATPase of yeast mitochondria

Abstract

Received 6 June 1977 1. Introduction Soluble ATPase of mitochondria is now an accepted component of the enzyme system catalyzing oxidative phosphorylation. According to the hypothesis of Boyer and Slater [ 1,2] a conformational transition of the enzyme protein is expected to be involved in ATP synthesis and the kinetic resolution of the ATPase reaction should give further insight into the mechanism of energy conservation in oxidative phosphorylation. The high purity of an ATPase preparation obtained in this laboratory [3] allowed to analyze the transient state kinetics of the hydrolytic reaction catalyzed by the enzyme. We here report that the catalytic process can be resolved into two steps, one of which is a slow conformational transition with a time constant of the order of 10 see-’ followed by a steady state hydrolysis with a turnover number of 200 set-‘. A preliminary report of this work was given elsewhere [4]. 2. Material and methods The soluble ATPase was prepared according to [3]. The specific activity of the enzyme was about 150 U/mg. The turnover number is given under the assumption of three active sites on the enzyme. The overall activity was determined in an ATP regenerating assay system as given elsewhere [ 51. For stopped flow experiments the enzyme protein was obtained from a stock solution and transferred to the appropriate buffer by spinning down the ammonium sulfate suspension of the enzyme, dissolving the pellet in the desired buffer and passing it through a Sephadex G-50 column preequilibrated with the same buffer. The enzyme was used immediately after this treatment in order to avoid significant activity loss occurring in the complete absence of ATP. Protein concentrations are based on a molecular weight of 400 000 [3]. ATP was obtained from Boehringer, Mannheim, E-ATP from Sigma and AMP-PNP (&y-imidoadenosine-5’-tri- phosphate) as well as phenolred from Serva, Heidelberg. All other chemicals were of the purest grade commercially available. The concentrations of ATP, E-ATP, AMP-PNP and phenolred were determined by absorbancy measurements in a Zeiss PMQ II photometer. Stopped flow experiments using phenolred as indicator were carried out with an instrument described elsewhere [6]. The proton release was evaluated by converting the obtained absorbancy changes of the indicator at 560 nm to proton concentrations liberated in the reaction using buffer-capacity of the system. The results were consistent with the end-point of the reaction as calculated from the total amount of ATP present in the reaction mixture. Experiments using the fluorescence change of E-ATP Mn(II) were performed with a Durrum stopped flow instrument connected to a Digital Storage oscilloscope (Nicolet Instrument Corporation Model 1074) allowing signal averaging. Here, the quenching effect of Mn’+- ions upon the fluorescence of E-ATP allows one to follow the reaction [7]. ATP forms a more stable complex with Mn”-ions than ADP, thus changing the amount of nucleotide present as manganese complex at constant manganese-concentrations.