Figure 1.

Mutation of Active Site Residues Alters the Specificity of Initial Attack on InsP₆ Substrate

(A–C) Products of reaction of native BtMinpp- (A), A31Y- (B), and R183D- (C) substituted enzyme with myo-inositol(1,[³²P]2,3,4,5,6)P₆ were resolved by Partisphere SAX HPLC. Mutated proteins were incubated at a concentration of 2.5 μg/ml and native protein at 0.25 μg/ml, with 1 mM InsP₆. The regions of the chromatogram in which InsP₃, InsP₄, and specific InsP₅ isomers elute are indicated in (A). For native enzyme (inset in A), reaction products were mixed with standards of myo-[2-³H]inositol (1,3,4,5,6)P₅ (InsP₅ [2-OH]), D/L- myo-[2-³H]inositol (1,2,4,5,6)P₅ (InsP₅ [1/3-OH]), and D/L- myo-[2-³H]inositol (1,2,3,4,5)P₅ (InsP₅ [4/6-OH]). Fractions, 0.25 min, were collected and radioactivity was estimated by scintillation counting; ³H, open circles; ³²P, filled circles. For R183D-substituted enzyme (inset in C), the reaction products were additionally mixed with standards of myo-[¹⁴C]InsP₅ [2-OH] and D/L- myo-[¹⁴C]InsP₅ [1/3-OH] and resolved on a Adsorbosphere SAX HPLC column. This column separates InsP₅ [2-OH] from InsP₅ [1/3-OH]. Fractions, 0.25 min, were collected and radioactivity estimated by scintillation counting: ¹⁴C, open squares; ³²P, filled circles.