Kinetic Investigation of Cooperativity in Coenzyme Binding by Transketolase Active Sites

M. V. Kovina¹, V. A. Selivanov², N. V. Kochevova¹, and G. A. Kochetov^1*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181; E-mail: kochetov@bac.genebee.msu.su

²Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia

^* To whom correspondence should be addressed.

Received October 31, 1997; Revision received November 27, 1997

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The two-step mechanism of coenzyme (thiamine diphosphate, ThDP) binding with two initially identical active sites of apotransketolase has been examined with a kinetic model. Cooperativity between sites in the primary ThDP binding and in the following conformational transition has been analyzed. The only reliable difference between sites is shown to be the tenfold difference in the backward rate constants of the conformational transition; this means that the cooperative interaction between sites takes place only after termination of both steps of ThDP binding in both sites.

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KEY WORDS: transketolase, thiamine diphosphate, kinetic modeling

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