Role of the covalent flavin linkage in monomeric sarcosine oxidase.
Monomeric sarcosine oxidase (MSOX) is a prototypical member of a recently recognized family of amine-oxidizing enzymes that all contain covalently bound flavin. Mutation of the covalent flavin attachment site in MSOX produces a catalytically inactive apoprotein (apoCys315Ala) that forms an unstable complex with FAD (K(d) = 100 muM), similar to that observed with wild-type apoMSOX where the complex is formed as an intermediate during covalent flavin attachment. In situ reconstitution of sarcosine oxidase activity is achieved by assaying apoCys315Ala in the presence of FAD or 8-nor-8-chloroFAD, an analogue with an approximately 55 mV higher reduction potential. After correction for an estimated 65% reconstitutable apoprotein, the specific activity of apoCys315Ala in the presence of excess FAD or 8-nor-8-chloroFAD is 14% or 80%, respectively, of that observed with wild-type MSOX. Unlike oxidized flavin, apoCys315Ala exhibits a high affinity for reduced flavin, as judged by results obtained with reduced 5-deazaFAD (5-deazaFADH(2)) where the estimated binding stoichiometry is unaffected by dialysis. The Cys315Ala.5-deazaFADH(2) complex is also air-stable but is readily oxidized by sarcosine imine, a reaction accompanied by release of weakly bound oxidized 5-deazaFAD. The dramatic difference in the binding affinity of apoCys315Ala for oxidized and reduced flavin indicates that the protein environment must induce a sizable increase in the reduction potential of noncovalently bound flavin (DeltaE(m) approximately 120 mV). The covalent flavin linkage prevents loss of weakly bound oxidized FAD and also modulates the flavin reduction potential in conjunction with the protein environment.
Hassan-Abdallah
Alshaimaa
A
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
Zhao
Guohua
G
Jorns
Marilyn Schuman
MS
D001059:Apoproteins / Q000737:chemistry / Q000235:genetics; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000737:chemistry* / Q000494:pharmacology; D009154:Mutation; D050879:Sarcosine Oxidase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics
C513553:8-nor-8-chloroflavine adenine dinucleotide; D001059:Apoproteins; D001426:Bacterial Proteins; D005182:Flavin-Adenine Dinucleotide; C023406:1-deaza-FAD; D050879:Sarcosine Oxidase; D003545:Cysteine