Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

摘要: Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago . contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes variety of reactions, such as halogenation, peroxidation epoxidation. The versatile catalytic activities CPO coupled with increasing demands for chiral synthesis have attracted an escalating interest in understanding mechanistic structural properties this enzyme. In order to better understand mechanisms CPO-catalyzed enantioselective reactions fine-tune chloroperoxidase, asparagine 74 (N74) located narrow substrate access channel was replaced bulky, nonpolar valine polar glutamine using site-directed mutagenesis. N74 mutants displayed significantly enhanced activity toward substrates compared wild-type result changes space polarity heme distal environment. More interestingly, showed dramatically decreased chlorination catalase but epoxidation consequence improved kinetic perfection introduced mutation reflected favorable k cat / K M these reactions. It also noted that N74V mutant capable decomposing cyanide, most notorious poison many hemoproteins, judged unique binding behavior potassium cyanide. Histidine 105 (H105) amino acid alanine H105 (H105A) possibly because environment loss hydrogen bonds between histidine glutamic 183. However, increased enantioselectivity observed bulky styrene derivatives. Furthermore, my study provides strong evidence proposed histidine/cysteine ligand switch providing experimental support structure 420-nm absorption maximum number carbon monoxide complexes heme-thiolate proteins. For NMR study, [dCPO(heme)] produced 90% deuterated growth medium excess precursors [dCPO(Phe)] grown same highly had been supplemented natural phenylalanine. To make complete proton assignments, spectroscopy has performed high-resolution characterization achieve unambiguous which allows important acids close active center be determined.