Abstract:
The role of polypeptide backbone interactions in 4-oxalocrotonate tautomerase (4OT) catalysis has been investigated using a combination of site-directed mutagenesis experiments with unnatural amino acids and quantum mechanical/molecular mechanical (QM/MM) calculations of the 4OT reaction mechanism. Energy barriers for the wild-type enzyme (wt-4OT) and for a 4OT analogue containing a backbone amide to ester bond mutation between Ile-7 and Leu-8 [(OL8)4OT] were determined by both theory and experiment. The amide to ester bond mutation in (OL8)4OT effectively deleted a putative hydrogen bonding interaction between the enzyme's polypeptide backbone and its substrate. Recent theoretical calculations for the 4OT reaction mechanism suggested that this hydrogen bonding interaction helps properly position the substrate in the active site [Cisneros, G. A., et al. (2003) J. Am. Chem. Soc. 125, 10384-10393]. Our experimental results for (OL8)4OT reveal that the energy barrier for the (OL8)4OT-catalyzed reaction was increased 1.8 kcal/mol over that of the wild-type enzyme. This increase was in good agreement with the 1.0 kcal/mol increase obtained from QM/MM calculations for this analogue. Our theoretical calculations further suggest the hydrogen bond deletion in (OL8)4OT results in a rearrangement of the substrate in the active site. In this rearrangement, an ordered water molecule loses its ability to stabilize the transition state (TS), and Arg-61 gains the ability to stabilize the TS. The predicted role of Arg-61 in (OL8)4OT catalysis was confirmed in kinetic experiments with an analogue of (OL8)4OT containing an Arg to Ala mutation at position 61.
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