QR Code
Uribe, S; Sampedro, JG; Munoz-Clares, RA (2002)

TREHALOSE-MEDIATED INHIBITION OF THE PLASMA MEMBRANE H+-ATPASE FROM KLUYVEROMYCES LACTIS: DEPENDENCE ON VISCOSITY AND TEMPERATURE

J BACTERIOL 184(16):4384-4391
full text

The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures. At 20degreesC, increasing concentrations of trehalose (0.2 to 0.8 M) decreased V-max and increased S-0.5 (substrate concentration when initial velocity equals 0.5 V-max), mainly at high trehalose concentrations (0.6 to 0.8 M). The quotient V-max/S-0.5 decreased from 5.76 mumol of ATP mg of protein(-1) min(-1) mM(-1) in the absence of trehalose to 1.63 mumol of ATP mg of protein(-1) min(-1) mM(-1) in the presence of 0.8 M trehalose. The decrease in V-max was linearly dependent on solution viscosity (1), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramer's theory for reactions in solution. In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted. In the absence of trehalose, increasing the temperature up to 40degreesC resulted in an exponential increase in V-max and a decrease in enzyme cooperativity (n), while S-0.5 was not modified. As temperature increased, the effect of trehalose on V-max decreased to become negligible at 40degreesC, in good correlation with the temperature-mediated decrease in viscosity. The trehalose-mediated increase in S-0.5 was similar at all temperatures tested, and thus, trehalose effects on V-max/S-0.5 were always observed. Trehalose increased the activation energy for ATP hydrolysis. Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.