The endo-oxabicyclic inhibitor binds to yeast CM in a similar way as it does to the distantly related CM from E. coli. The inhibitor-binding mode supports a mechanism by which polar sidechains of the enzyme bind the substrate in the pseudo-diaxial conformation, which is required for catalytic turnover. A lysine and a protonated glutamate sidechain have a critical role in the stabilization of the transition state of the pericyclic reaction. The allosteric transition from T-->R state is accompanied by a 15 degrees rotation of one of the two subunits relative to the other (where 0 degrees rotation defines the T state). This rotation causes conformational changes at the dimer interface which are transmitted to the active site. An allosteric pathway is proposed to include residues Phe28, Asp24 and Glu23, which move toward the activesite cavity in the T state. In the presence of the transition-state analogue a super R state is formed, which is characterised by a 22 degrees rotation of one subunit relative to the other.

The crystal structure of an allosteric chorismate mutase, the Thr-226 -B le mutant, from yeast Saccha-romyces cerevisiae has been determined to 2.2-A resolution by using the multiple isomorphous replacement method. Solventflafening and electron-density modification were applied for phase improvement. It has been proposed that both enzymatic and nonenzymatic rearrangement of chorismate to prephenate is a concerted reaction that proceeds via a transition state with chair-like geometry (3-6). The enzymatic reaction is distinctive among known enzymes in that it catalyzes a pericyclic process. The recently published crystal structure of monofunctional chorismate mutase from B. subtilis shows that no functional group from the protein is available for direct involvement in the catalysis (7). The two-million-fold rate enhancement is thus achieved by selection of the active conformation and stabilization of the transition state by the enzyme.The structural data on the catalytic antibody with low chorismate mutase activity (8) also supports the conclusion that the catalysis ofthe isomerization of chorismate proceeds by stabilizing the same pericyclic transition state that occurs in the uncatalyzed thermal reaction, rather than by providing alternative pathways to the product.Yeast chorismate mutase is a monofunctional dimer oftwo 30-kDa polypeptides encoded by the AR07 gene (9). Biochemical data showed that in the dimer there are two substrate-binding sites, two activator-binding sites, and two inhibitor-binding sites that may or may not be distinct from the activator sites. Kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate. This cooperativity is lost in the presence of tryptophan. The product of one of the AR07 mutant alleles, with a single substitution of Ile-226 (T226I), has been characterized as a constitutively activated chorismate mutase that does not respond to activation by tryptophan or inhibition by tyrosine. As a relatively small protein in the allosteric family, yeast chorismate mutase provides an ideal model system for exploring the detailed mechanisms of allosteric regulation as well as to examine the unique catalysis. We describe here the crystal structure of the mutant chorismate mutase, T2261, from yeast S. cerevisiae. § In contrast to the available structure of chorismate mutase from B. subtilis which exists as a homotrimer, the yeast protein is a dimer that has a completely different folding topology. In addition, the binding sites for tryptophan in the structure were identified. MATERIALS AND METHODSThe mutant enzyme was isolated, purified, and characterized by R.G., G.S., and G.B., and the structure determination was made by Y.X. and W.N.L., as described below.The crystallization of the protein was done by using the hanging-drop method, as described earlier (10). The space group was established as P6 or P65, and the unit cell parameters are a = b = 95.8 , c = 157.9 A, a =p = 90°, and y = 120°. Heavy-atom derivatives were prepared by soaking the c...

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase͞prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

The crystal structure of the tyrosine-bound T

Acetobacterium woodii, Acetohalobium arabaticum, Clostridium formicoaceticum, and Sporomusa silvacetica were found to contain carbonic anhydrase (CA). Minimal to no CA activity was detected in Moorella thermoautotrophica, Moorella thermoacetica subsp. "pratumsolum," Sporomusa termitida, and Thermoanaerobacter kivui. Of the acetogens tested, A. woodii had the highest CA specific activity, approximately 14 U mg of protein ؊1 , in extracts of either glucose-or H 2 -CO 2 -cultivated cells. CA of A. woodii was cytoplasmic and was purified approximately 300-fold to a specific activity of 5,236 U mg of protein ؊1