The Enzymology of the Monooxygenase Domain of MICAL-2.

Mechanistic Enzymology;The Mechanism of MICAL;a Flavin-dependent Monooxygenase That Depolymerizes Actin;Biological Chemistry;Science;Biological Chemistry

McDonald, Claudia AlejandraFierke, Carol A. ;

University of Michigan

关键词: Mechanistic Enzymology;    The Mechanism of MICAL;    a Flavin-dependent Monooxygenase That Depolymerizes Actin;    Biological Chemistry;    Science;    Biological Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/97851/claudale_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Molecules interacting with CasL (MICALs) are large multi-domain flavin-dependent monooxygenases that use redox chemistry to cause actin to depolymerize. Little enzymology has been reported for MICAL-2, an enzyme vital for the proliferation of prostate cancer cells. The monooxygenase domains of MICALs resemble aromatic hydroxylases, but their substrate is the sulfur of a methionine of actin. In order to determine how closely MICAL-2 conforms to the aromatic hydroxylase paradigm, the half-reactions were studied in stopped-flow experiments.The enzyme had a strong preference for NADPH over NADH and is specific for the proR hydride (like other aromatic hydroxylases), as evidenced by a 4.8-fold kinetic isotope effect. In the absence of actin, NADPH reduces the flavin relatively slowly; actin speeds that reaction significantly.Stopped-flow experiments were also used to monitor the oxidation of the enzyme-bound reduced FAD in the absence and in the presence of f-actin, the putative substrate to be oxygenated. In the absence of actin, flavin was oxidized in two phases because there are two populations of enzyme. The comparison of increases in absorbance at 450 nm, an indicator of flavin oxidation, and 550 nm, an indicator of flavin conformation, suggests that one form reacts relatively fast with oxygen after the flavin moves from the ;;in” to the ;;out” conformation. The other form of the enzyme oxidizes slower, and the absorbance at 550 nm increases even slower, suggesting that this form reacts when the flavin is ;;in” and subsequent movement to the ;;out” conformation is slow.When f-actin was included in reaction mixtures, the flavin hydroperoxide and the flavin hydroxide were observed. The formation of the hydroperoxide was faster by an order of magnitude than the reaction of actin-free MICAL with oxygen. At the concentrations of actin used in these experiments (tens of micromolar), about half the reactive hydroperoxide intermediate eliminated hydrogen peroxide. The enzyme that successfully oxygenated actin formed the flavin hydroxide intermediate and, after eliminating water, formed oxidized enzyme. These results show that monoMICAL-2 behaves like a typical aromatic hydroxylase in most of its reactions. The striking difference is the ability to oxygenate a non-aromatic nucleophile