期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：5
页码：1183-1188
DOI：10.1073/pnas.0304369101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the KM (the [ATP] required for half-maximal speed) was 28 {+/-} 1 {micro}M, and the maximum speed was 904 nm/s. Pi behaved as a competitive inhibitor with KI = 9 {+/-} 1 mM. ADP behaved approximately as a competitive inhibitor with KI = 35 {+/-} 2 {micro}M. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which Pi is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release Pi before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes Pi release from the other.
关键词：crossbridge cycle ; motor protein ; chemomechanical coupling
