CRYSTAL-STRUCTURES AND MECHANISM OF 6-PHOSPHO-BETA-GALACTOSIDASE FROMLACTOCOCCUS-LACTIS

The initial structural model of 6-phospho-beta-galactosidase from Lact ococcus lactis was refined to an R-factor of 16.4% (R-free = 23.6%) to 2.3 Angstrom resolution (1 Angstrom = 0.1 nm), and the structures of three other crystal forms were solved by molecular replacement. The fo ur structural models are essentially identical. The catalytic center o f the enzyme is approximately at the mass center of the molecule and c an only be reached through a 20 Angstrom long channel, which is observ ed with an ''open'' or ''closed'' entrance. The closed entrance is pro bably too small for the educt lactose-6-phosphate to enter, but large enough for the first product glucose to leave. Among the presented str uctures is a complex between an almost inactive mutant and the second product galactose-6-phosphate, which is exclusively bound at side-chai ns. A superposition (onto the native enzyme) of galactose-6-phosphate as bound to the mutant suggests the geometry of a postulated covalent intermediate. The binding mode of the educt was modeled, starting from the bound galactose-6-phosphate. A tightly fixed tryptophan is used a s a chopping-board for splitting the disaccharide, and several other a romatic residues in the active center cavity are likely to participate in substrate transport/binding. (C) 1997 Academic Press Limited.