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Biochemistry. 2012 May 8;51(18):3933-40. doi: 10.1021/bi201881p. Epub 2012 Apr 24.

Catalysis by a de novo zinc-mediated protein interface: implications for natural enzyme evolution and rational enzyme engineering.

Author information

1
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.

Abstract

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein-protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 10(5) and a k(cat)/K(M) of 630 M(-1) s(-1) and 4-nitrophenyl phosphate with a rate acceleration of 10(4) and a k(cat)/K(M) of 14 M(-1) s(-1). These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein-protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

PMID:
22510088
PMCID:
PMC3348550
DOI:
10.1021/bi201881p
[Indexed for MEDLINE]
Free PMC Article

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