COUPLING OF KINESIN STEPS TO ATP HYDROLYSIS

A key goal in the study of the function of ATP-driven motor enzymes is to quantify the movement produced from consumption of one ATP molecul e(1-3). Discrete displacements of the processive motor kinesin along a microtubule have been reported as 5 and/or 8 mn (refs 4, 5). However, analysis of nanometre-scale movements is hindered by superimposed bro wnian motion, Moreover, because kinesin is processive and turns over s tochastically, some observed displacements must arise from summation o f smaller movements that are too closely spaced in time to be resolved , To address both of these problems, we used light microscopy instrume ntation(6) with low positional drift (<39 pm s(-1)) to observe single molecules of a kinesin derivative moving slowly (similar to 2.5 nm s(- 1)) at very low (150 nM) ATP concentration, so that ATP-induced displa cements were widely spaced in time, This allowed increased time-averag ing to suppress brownian noise (without application of external force( 4,5)), permitting objective measurement of the distribution of all obs erved displacement sizes, The distribution was analysed with a statist ics-based method which explicitly takes into account the occurrence of unresolved movements, and determines both the underlying step size an d the coupling of steps to ATP hydrolytic events, Our data support a f undamental enzymatic cycle for kinesin in which hydrolysis of a single ATP molecule is coupled to a step distance of the microtubule protofi lament lattice spacing of 8.12 nm (ref. 7). Step distances other than 8 nm are excluded, as is the coupling of each step to two or more cons ecutive ATP hydrolysis reactions with similar rates, or the coupling o f two 8-nm steps to a single hydrolysis. The measured ratio of ATP con sumption rate to stepping rate is invariant over a wide range of ATP c oncentration, suggesting that the 1 ATP to 8 nm coupling inferred from behaviour at low ATP can be generalized to high ATP.