4-Oxalocrotonate tautomerase (EC 5.3.2-; 4-OT), a hexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids, converting unconjugated ketones to the conjugated isomers via a dienolic intermediate. The recently solved crystal structure of an isozyme of 4-OT suggests that the amino-terminal proline is the catalytic base [Subramanya, H. S., Roper, D. I., Dauter, Z., Dodson, E. J., Davies, G. J., Wilson, K. S., & Wigley, D. B. (1996) Biochemistry 35, 792-802]. In support of this proposed role, we have found that the active-site-directed irreversible inhibitor 3-bromopyruvate (3-BP) blocks the amino terminus of 4-OT to Edman degradation and results in the disappearance of the 15N resonance of Pro-1 (delta = 49.2 ppm at pH 6.40 and 42 degrees C) in the 15N NMR spectrum of uniformly 15N-labeled 4-OT. Furthermore, covalent bonding between a 15N resonance of 4-OT and the methylene carbon of the reduced, 3-(13)C-labeled lactyl adduct derived from [3-(13)C]-bromopyruvate was then directly demonstrated using two heteronuclear NMR methods, an 1H-(13)C HSQC experiment and a novel inverse correlation experiment which we call H(C)N. The chemical shift of the modified 15N resonance (delta = 86.5 ppm) is consistent with that of an alkylated and cationic, amino-terminal proline. Affinity labeling with 2-(14)C-labeled bromopyruvate indicates that the ultimate stoichiometry of modification is I equiv of 3-BP per 4-OT monomer. However, an analysis of the residual enzyme activity after differing extents of fractional modification with 3-BP indicates that modification of three active sites per hexamer abolishes essentially all activity of the hexamer. Thus, 4-OT exhibits half-of-the-sites stoichiometry with 3-BP. Finally, the pH dependence of kinact/KI for affinity labeling by 3-BP yields a pKa value of 6.7 +/- 0.3, in reasonable agreement with the pKa values found for kcat/KM for the non-sticky substrate 2-hydroxy-2,4-pentadienoate and by direct NMR titration of Pro-1 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., & Whitman, C. P. (1996) Biochemistry 35, 814-823]. These results strongly implicate the amino-terminal proline as the general-base catalyst on 4-OT.

Creatine kinase (CK; EC 2.7.3.2) catalyzes the reversible conversion of creatine and MgATP to phosphocreatine and MgADP. In the absence of an X-ray crystal structure, we have used the sequence homology of creatine kinases and other guanidino kinases from a variety of sources to identify the conserved histidine residues in rabbit muscle CK, as well as to try to pinpoint a reactive histidine that has been implicated in the active site. This residue has been proposed to act as a general acid/base catalyst assisting in the phosphoryl transfer mechanism [Cook et al. (1981) Biochemistry 20, 1204-1210]. There are 17 histidine residues in rabbit muscle CK, and of these, only five have been conserved in all guanidino kinase sequences published to date [Mühlebach et al. (1994) Mol. Cell. Biochem. 133, 245-62]. In rabbit muscle CK, these residues are H96, H105, H190, H233, and H295. We have carried out site-specific mutagenesis of these five histidine residues, replacing each with an asparagine. Each of these mutants exhibited enzymatic activity but to varying degrees. The H105N, H190N, and H233N mutants displayed specific activities similar to that of the wild-type enzyme. H96N has high activity, but appears to be quite unstable, losing catalytic activity upon cell lysis by sonication and/or chromatographic steps involved in purification. H295N shows a significantly reduced catalytic activity relative to the native enzyme, due to marked decreases in kcat and the affinities for both substrates. Each of the five mutants is inactivated by diethyl pyrocarbonate (DEP), and inactivation is reversible upon incubation with hydroxylamine. However, only H295N shows a dramatically reduced rate of inactivation relative to native CK, consistent with H295 being the residue modified by DEP in the native enzyme. These intriguing results indicate that four of the conserved histidines (H96, H105, H295, and H233) are not essential for activity, and while H295 may be at the active site of CK, it is unlikely to play the role of a general acid/base catalyst.

Isoelectric focusing separation of recombinant rabbit muscle creatine kinase (CK) and its 282Cys-->282Ser mutant shows the presence of three and two isoforms, respectively, that exhibit equivalent enzymatic activity. Electrospray ionization coupled with Fourier-transform mass spectrometry (10(5) resolving power) of both CKs indicates that their major components are within +/- 2 Da of the M(r) value predicted from the cDNA sequences of these mixtures. Dissociation of (M + nH)n+ gives no evidence that the components of either CK are isomers; the masses of the 51 fragment ions correlate completely (+/- 1 Da) with the values predicted from the cDNA sequence and confirm the identities of 21 of the 380 amino acids and the 282Cys-->282Ser replacement in the mutant. The results are consistent with one or two steps of post-translational amidation/deamidation (NH2-->OH, 16 Da-->17 Da), each of which would produce only a 1 Da difference in M(r), with the fragment masses indicating that at least one modification occurs between residues 212 and 282.

We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11A␣ and 11A␤. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO 4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11A␣ was determined to be identical to 11A. 11A␤ is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO 4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11A␣ and 11A␤ subtypes. The polysaccharide (PS)2 capsule of Streptococcus pneumoniae is recognized as the most important virulence factor of pneumococci. It is expressed by almost all pathogenic pneumococci and has been shown to increase the virulence of a pneumococcal strain by more than a million-fold in an animal model system (1). The capsule shields pneumococci from the host phagocytes, and its shielding capacity can be neutralized when the host produces antibodies to the capsule and to fix complement on pneumococci. Thus, the capsular PS is used as the antigen in all pneumococcal vaccines that are clinically used. Because of their impact on human health, pneumococcal capsules have been extensively investigated both serologically, genetically, and biochemically. Many years of serological studies have identified 91 serologically distinct capsule types (2, 3). Recently, the nucleotide sequences of the capsule gene loci of all 91 different capsule types have been determined (4, 5).With the use of monoclonal antibodies, serologic heterogeneity was noted recently among pneumococcal isolates that were typed as 11A using the quellung reaction (6). To avoid confusion, we have provisionally named the common variant 11A␣ and the less common variant 11A␤. Because 11A PS is a component of the 23-valent pneumococcal PS vaccine (7), it is important to understand the chemical basis of the serological heterogeneity among pneumococci that are typed as serotype 11A. However, there is only limited information on structure of pneumococcal serotype 11A PS, and the structure has not been examined with modern tools. Forty years ago, the structural model for S. pneumoniae serotype 11A PS was proposed to be a linear polymer containing D-glucose, D-galactose, glycerol, phosphate, and O-acetyl groups in the molar ratio of 2:2:1:1:2 based upon analysis of chemically modified PS (8). About 20 years ago, Richards et al. (9,10) proposed the model for 11A PS based on both NMR and chemical degradation studies. They proposed a linear repeating unit structure with four monosaccharides, a pendant glycerol phosphate moiety, and 2 mol of acetate as described in Structure ...