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Volume 272, Number 4, Issue of January 24, 1997 pp. 2218-2222
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

(Received for publication, May 30, 1996, and in revised form, October 7, 1996)

Masayuki Nakanishi , Kazuya Matsuura , Hiroyuki Kaibe , Nobutada Tanaka ^¶ , Takamasa Nonaka ^¶ , Yukio Mitsui ^¶ and Akira Hara

From the  Biochemistry Laboratory, Gifu Pharmaceutical University, Gifu 502, Japan and the ^¶ Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-21, Japan

Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, exhibits coenzyme specificity for NADP(H) over NAD(H). Crystal structure of the enzyme-NADPH complex shows that Thr-38 interacts with the 2-phosphate of NADPH and occupies the position spatially similar to an Asp residue of the NAD(H)-dependent SDRs that hydrogen-bonds to the hydroxyl groups of the adenine ribose of the coenzymes. Using site-directed mutagenesis, we constructed a mutant mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D), and we compared kinetic properties of the mutant and wild-type enzymes in both forward and reverse reactions. The mutation resulted in increases of more than 200-fold in the K_m values for NADP(H) and decreases of more than 7-fold in those for NAD(H), but few changes in the K_m values for substrates or in the k_cat values of the reactions. NAD(H) provided maximal protection against thermal and urea denaturation of T38D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the single mutation converted the coenzyme specificity from NADP(H) to NAD(H). Calculation of free energy changes showed that the 2-phosphate of NADP(H) contributes to its interaction with the wild-type enzyme. Changing Thr-38 to Asp destabilized the binding energies of NADP(H) by 3.9-4.5 kcal/mol and stabilized those of NAD(H) by 1.2-1.4 kcal/mol. These results indicate a significant role of Thr-38 in NADP(H) binding for the mouse lung enzyme and provide further evidence for the key role of Asp at this position in NAD(H) specificity of the SDR family proteins.

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