CRYSTAL-STRUCTURE OF THE K12M G15A TRIOSEPHOSPHATE ISOMERASE DOUBLE MUTANT AND ELECTROSTATIC ANALYSIS OF THE ACTIVE-SITE/

The crystal structure of the yeast triosephosphate isomerase (TIM) dou ble mutant K12M/G15A has been solved to 2 Angstrom by X-ray diffractio n, and the effects of changing the positively charged lysine to the ne utral methionine have been analyzed. The mutant enzyme was crystallize d in the presence of the tight-binding inhibitor phosphoglycolohydroxa mate, under standard conditions for obtaining crystals of the enzyme-i nhibitor complex. The crystals obtained were of the same crystal form as the unliganded wild-type enzyme. The three-dimensional structure co nfirms that the Lys-12 to Met mutation prevents the enzyme from bindin g substrate and reveals that the reason is electrostatic and not steri c. 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 elect rostatic potential at the active site of the mutant enzyme is, however , very different from that of the wild type. It has been postulated pr eviously that Lys-12 may play a role in stabilizing the negative charg e in the transition state. This K12M/G15A structure suggests that the active-site Lys, which is strictly conserved, is required for TIM to b e able to bind its dianionic substrate.