Sarcosine oxidase: structure, function, and the application to creatinine determination.

Area of Science:

• Biochemistry
• Enzymology
• Clinical Chemistry

Background:

• Creatinine determination is vital in clinical diagnostics.
• The Jaffé reaction for creatinine is prone to interferences.
• Enzymatic methods, utilizing sarcosine oxidase, offer improved accuracy.

Purpose of the Study:

• To investigate the kinetic and structural properties of Corynebacterium enzyme, a key sarcosine oxidase.
• To understand the role of noncovalently and covalently bound flavin adenine dinucleotide (FAD) in enzyme activity.
• To explore methods for enhancing enzyme specificity in creatinine assays.

Main Methods:

• Studied the kinetic properties of Corynebacterium sarcosine oxidase.
• Analyzed the enzyme's structure, including its four non-identical subunits (A, B, C, D).
• Employed chemical modification using iodoacetamide to probe FAD binding sites and enzyme function.

Main Results:

• Identified distinct noncovalently and covalently bound FADs within the enzyme complex.
• Established that covalently bound FAD is associated with subunit B.
• Chemical modification revealed the amino acid sequence around the non-covalently bound FAD and altered enzyme function.

Conclusions:

• The rate of sarcosine oxidation is dependent on the redox states of both bound FADs.
• Chemical modification selectively inactivated the covalently bound FAD, leaving only the noncovalently bound FAD active.
• This modification enhances the enzyme's utility in specific creatinine assays by ensuring reliance on a single FAD cofactor.