Substrate channeling

被引:161
作者
Spivey, HO
Ovádi, J
机构
[1] Oklahoma State Univ, Dept Biochem & Mol Biol, Stillwater, OK 74078 USA
[2] Hungarian Acad Sci, Biol Res Ctr, Inst Enzymol, H-1518 Budapest, Hungary
基金
美国国家科学基金会;
关键词
D O I
10.1006/meth.1999.0858
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Substrate channeling is the process in which the intermediate produced by one enzyme is transferred to the next enzyme without complete mixing with the bulk phase, This process is equivalent to a microcompartmentation of the intermediate, although classic diffusion occurs simultaneously to varying extents in many of these cases. This microcompartmentation and other factors of channeling provide many potential biological advantages. Extensive examples of channeling can be found in the cited reviews. The choice of methods to detect and characterize substrate channeling depends extensively on the type of enzyme associations involved, the constants of the system, and, to some extent, the mechanism of channeling. Thus it is important to distinguish stable, dynamic, and catalytically induced enzyme associations as well as recognize different mechanisms of substrate channeling We discuss the principles, experimental details, and limitations and precautions of five rather general methods. These use measurements of transient times, isotope dilution or enhancement, competing reaction effects, enzyme buffering kinetics, and transient-state kinetics. These encompass methods applicable to studies in vitro, in situ, and in vivo. None of these methods is applicable to all systems. They are also susceptible to artifacts without proper attention to precautions. Transient-state kinetic methods clearly excel in elucidating molecular mechanisms of channeling. However, they are often not the best method for initial detection and characterization of the process and they are not applicable to many complex systems. Several other methods that have been successful in indicating substrate channeling are briefly described. (C) 1999 Academic Press.
引用
收藏
页码:306 / 321
页数:16
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