Studies on milk xanthine dehydrogenase.

Milk xanthine oxidase (MXO) is a metalloflavoprotein which converts xanthine to urate. The dimeric enzyme contains one FAD, two (2Fe-2S) centers, and one molybdopterin cofactor per subunit. Xanthine is oxidized to urate by the molybdopterin, and reducing equivalents are passed to the flavin, which is reoxidized by oxygen. By purifying the protein in the presence of the thiol reducing agent dithiothreitol, we have isolated the protein as a dehydrogenase form (MXDH) which reacts well with NAD and poorly with oxygen. Presumably, reducing disulfide bonds within the enzyme causes a conformational change, creating a binding site for NAD. Studies reported in this thesis examine the differences between the MXO and MXDH forms of the protein. The dehydrogenase form can be converted back to the oxidase form by incubation with the disulfide forming reagent 4,4$\sp\prime$-dithiopyridine, indicating that conversion of MXO to MXDH occurs through the reduction of disulfide bonds within the protein. MXDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 min$\sp{-1}$ at pH 7.5, 25$\sp\circ$C, with a Km of $<$1$\mu$M for xanthine and a Km of $\approx$7$\mu$M for NAD, but has little O$\sb2$-dependent activity. Rapid reaction studies show that the reduction of the enzyme by xanthine is rate-limiting in catalysis. Evidence that the two enzyme forms have different flavin environments was shown by studies in which the native FAD was substituted with the artificial flavin 8-SH-FAD. MXDH binds this flavin in its neutral form, elevating the pK of 8-SH-FAD by 5 pH units, while MXO binds this flavin in its anionic form at pH 7.5. A difference was found between the redox potentials of the native flavin in MXO and MXDH. The FAD/FADH$\sp\cdot$ potentials are similar in both forms of the protein, but the FADH$\sp\cdot$/FADH$\sb2$ couple is 180 mV lower in MXDH than in MXO. This provides stabilization of the neutral flavin semiquinone in MXDH and lowers the midpoint of the flavin to a value nearer that of the NAD/NADH couple, enhancing the enzyme's ability to reduce NAD during catalysis.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103959/1/9423213.pdfDescription of 9423213.pdf : Restricted to UM users only