Crystal structure of the K12M/G15A triosephosphate isomerase double mutant and electrostatic analysis of the active site.

Abstract

The crystal structure of the yeast triosephosphate isomerase (TIM) double mutant K12M/G15A has been solved to 2 A by X-ray diffraction, and the effects of changing the positively charged lysine to the neutral methionine have been analyzed. The mutant enzyme was crystallized in the presence of the tight-binding inhibitor phosphoglycolohydroxamate, under standard conditions for obtaining crystals of the enzyme-inhibitor complex. The crystals obtained were of the same crystal form as the unliganded wild-type enzyme. The three-dimensional structure confirms that the Lys-12 to Met mutation prevents the enzyme from binding substrate and reveals that the reason is electrostatic and not steric. The substrate-binding loop is in its open position and the Met side chain points away from the active site. Overall, the mutant structure is very similar to that of the wild-type unliganded enzyme. The electrostatic potential at the active site of the mutant enzyme is, however, very different from that of the wild type. It has been postulated previously that Lys-12 may play a role in stabilizing the negative charge in the transition state. This K12M/G15A structure suggests that the active-site Lys, which is strictly conserved, is required for TIM to be able to bind its dianionic substrate.