Determinants of Specificity |
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........ The superposition of NADP-specific IDH with NADP bound (2'P, H-bonds) on NAD-specific Hurely & Dean. 1994. Structure 2:1007-1016. |
The advent of site directed mutagenesis brought with it an ability to test hypotheses about the relations between protein structure and function. We use site directed mutagenesis to test hypotheses concerning the substrate specificity and catalytic mechanisms of enzymes in the IDH superfamily. |
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| We were the first to successfully engineer an enzyme from being NADP-specific to being NAD-specific. We showed that six amino acid replacements in the active site of IDH were sufficient to cause a 1.4 million-fold change in specificity. | ||
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| Chen, Greer & Dean. 1995. PNAS 92:11666-11670. | ||
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| We were the first to engineer the secondary structure of an active site to elicit a desired change in specificity, this time from NAD to NADP in IMDH. | ||
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| Chen, Greer & Dean. 1996. PNAS 93:12171-12176. | ||
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| Successfully inverting the specificities of two homologous enzymes shows that hundreds of other mutations accumulated during the course of 4 billion years of evolution have negligible impacts on NADP/NAD use. | ||
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| Hence, functional evolution may proceed through a few mutations of massive effect. | ||
