Inactivation of Monomeric Sarcosine Oxidase by a Suicide Substrate
EMSL Project ID
2073
Abstract
We have recently determined the kinetic mechanism of inactivation of monomeric sarcosine oxidase by N-(cyclopropyl)glycine (CPG), a sarcosine analog which acts as a suicide substrate and covalently modifies the flavin prosthetic group of the enzyme. We are now working on determining the chemical mechanism of inactivation. The modified flavin in CPG-modified MSOX is unstable since it cycles back to unmodified flavin via a 1,5-dihydroFAD intermediate. The modified flavin can be stabilized by further reduction with sodium borohydride. In collaboration with Dr. Scott Mathews we have recently crystallized the borohydride-treated CPG-modified MSOX. A data set has been collected at 1.85 A and is currently under analysis. The molecular weight of the borohydride-reduced CPG-modified flavin would provide valuable information with respect to interpreting the new electron density near the flavin. The modified flavin in this construct is stable upon denaturation with guanidine hydrochloride in the sense that it does not revert to unmodified oxidized flavin but small spectral changes are observed over a period of hours after denaturation. So the ideal mass spectral approach might be to get the molecular mass of the intact enzyme using the 7 tesla FTICR, analogous to our studies a couple of years ago with the much larger and more complex heterotetrameric sarcosine oxidase.
Project Details
Project type
Exploratory Research
Start Date
2000-11-03
End Date
2002-02-28
Status
Closed
Released Data Link
Team
Principal Investigator