| heal.abstract |
Oxalate oxidase (OXO) was chemically modified using amino acid-specific reagents. The modification reactions were monitored spectrophotometrically, to follow the progress of labeling, and catalytically, to assess the effect of labeling on the enzyme function. The enzyme does not bear arginines essential for activity, since 2,3-butanedione and cyclohexanodione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Incubation of urea-pretreated OXO with N-acetylimidazole leads to labeling all 10 tyrosines without affecting the enzyme activity, thus suggesting that OXO does not have tyrosines essential for activity. However, OXO modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide followed by kinetic analysis, leads to the conclusion that the enzyme possesses one carboxylate essential for activity. When using the modifier 2,4,6- trinitrobenzene sulfonic acid (TNBS), while 28 of the total 45 lysines are labeled within 3 h (the first 5 reacting lysines of the homopentametic enzyme are modified at a faster rate than the others), the enzyme rapidly loses 90% of its activity in the first 2 min, a period during which only one lysine is being labeled. Complete enzyme inactivation with TNBS is observed after approximately 8 min, when 5 lysines are being labeled. The modification of the first lysine also triggers the dissociation of native OXO to its subunits (after SDS-PAGE analysis), a phenomenon not observed with the other modifiers. These findings indicate that OXO bears a lysine per monomer, essential for enzyme activity. When using 5,5-dithio-bis-(2-nitrobenzoic)acid to determine the number of disulfide bonds, in the presence of NaBH4, 10 sulfhydryls are determined, but in the absence of reducing agent, none are determined. Further, chloro-mercuribenzoate does not inactivate OXO but β- mercaptoethanol does. Therefore, the sulfhydryls in OXO are not free but form disulfide bonds essential for activity. Furthermore, the metallo-chelating agents HgCl2 and 8-hydroxychinolin inactivate the enzyme, suggesting that barley root oxalate oxidase is a metalloenzyme. It is possible that the metal(s) are involved in the oxidative mechanism since the enzyme does not bear prosthetic groups such as FAD and FMN. |
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