Search

Tautomerase superfamily (TSF) members are constructed from a single β−α−β unit or two consecutively joined β−α−β units. This pattern prevails throughout the superfamily consisting of more than 11,000 members where homo- or heterohexamers are localized in the 4-oxalocrotonate tautomerase (4-OT) subgroup and trimers are found in the other four subgroups. One exception is a subset of sequences that are double the length of the short 4-OTs in the 4-OT subgroup, where the coded proteins form trimers. Characterization of two members revealed an interesting dichotomy. One is a symmetric trimer, whereas the other one is an asymmetric trimer. One monomer is flipped 180° relative to the other two monomers so that three unique protein-protein interfaces are created that are composed of different residues. A bioinformatics analysis of the fused 4-OT subset shows a further division into two clusters with a total of 133 sequences. The analysis showed that members of one cluster (86 sequences) have more salt bridges if the asymmetric trimer forms, whereas the members of the other cluster (47 sequences) have more salt bridges if the symmetric trimer forms. This hypothesis was examined by the kinetic and structural characterization of two proteins within each cluster. As predicted, all four proteins function as 4-OTs, where two assemble into asymmetric trimers (designated R7 and F6) and two form symmetric trimers (designated W0 and Q0). These findings can be extended to the other sequences in the two clusters in the fused 4-OT subset, thereby annotating their oligomer properties and activities.

The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature's strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the alpha-proton of a carboxylate substrate that is coordinated to an essential Mg(2+). The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the beta-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologous MLEs that catalyzes anti-cycloisomerization has been identified. Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, gi 70731221 ; anti, Mycobacterium smegmatis, gi 118470554 ) document that the conserved Lys at the end of the second beta-strand in the (beta/alpha)(7)beta-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of "pseudoconvergent" evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction.

NagA catalyzes the hydrolysis of N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-D-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate inmore » conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the {alpha}-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.« less

A kinetic study of the reversible deprotonation of methylnitroacetate (4H) by primary aliphatic amines, secondary alicyclic amines, hydroxide ion, and water in water at 25 degrees C and in 50% DMSO/50% water (v/v) at 20 degrees C is reported. Intrinsic rate constants, k0, determined by extrapolation or interpolation of Brønsted plots have been determined. In comparison to proton transfers involving other nitroalkanes, the intrinsic rate constants for 4H are exceptionally high; for example, log k0 for the reaction of 4H with secondary alicyclic amines in water (1.22) is 1.81 log units higher than log k0 for nitromethane (-0.59), while in 50% DMSO/50% water, log k0 for 4H (2.44) is 1.71 log units higher than that for nitromethane (0.73). A general discussion of the factors affecting intrinsic rate constants of proton transfer from nitroalkanes is presented; it provides the context for an understanding as to why k0 is so high for the proton transfers from 4H. The correlation between intrinsic rate constants for the addition of nucleophiles to alkenes of the type R'R' 'C=CXY and the intrinsic rate constants of proton transfers from carbon acids of the type H2CXY is also discussed as a general proposition as well as with specific reference to the Ph(SMe)C=C(NO2)CO2Me/H2C(NO2)CO2Me pair.

A spectroscopic and kinetic study of the reaction of methyl beta-methylthio-alpha-nitrocinnamate (4-SMe) with morpholine, piperidine, and hydroxide ion in 50% DMSO/50% water (v/v) at 20 degrees C is reported. The reactions of 4-SMe with piperidine in a pH range from 10.12 to 11.66 and those with morpholine at pH 12.0 are characterized by two kinetic processes when monitored at lambdamax (364 nm) of the substrate, but by only one process when monitored at lambdamax (388) nm of the product. The rate constants obtained at 388 nm were the same as those determined for the slower of the two processes at 364 nm. These rate constants refer to product formation, whereas the faster process observed at 364 nm is associated with the loss of reactant to form an intermediate. In contrast, for the reaction of 4-SMe with morpholine at pH 8.62 the rates of product formation and disappearance of the substrate were the same, i.e., there is no accumulation of an intermediate. Likewise, the reaction of 4-SMe with OH- did not yield a detectable intermediate. The factors that allow the accumulation of intermediates in certain SNV reactions but not in others are discussed in detail, and structure-reactivity comparisons are made with reactions of piperidine and morpholine with other highly activated vinylic substrates.

The study of mechanistically diverse enzyme superfamilies-collections of enzymes that perform different overall reactions but share both a common fold and a distinct mechanistic step performed by key conserved residues-helps elucidate the structure-function relationships of enzymes. We have developed a resource, the structure-function linkage database (SFLD), to analyze these structure-function relationships. Unique to the SFLD is its hierarchical classification scheme based on linking the specific partial reactions (or other chemical capabilities) that are conserved at the superfamily, subgroup, and family levels with the conserved structural elements that mediate them. We present the results of analyses using the SFLD in correcting misannotations, guiding protein engineering experiments, and elucidating the function of recently solved enzyme structures from the structural genomics initiative. The SFLD is freely accessible at http://sfld.rbvi.ucsf.edu.

A kinetic study of the acid-catalyzed loss of alkoxide and thiolate ions from alkoxide and thiolate ion adducts, respectively, of benzylidene Meldrum's acid (1-H), methoxybenzylidene Meldrum's acid (1-OMe), and thiomethoxybenzylidene Meldrum's acid (1-SMe) is reported. The reactions appear to be subject to general acid catalysis, although the catalytic effect of buffers is weak and the bulk of the reported data refers to H(+)-catalysis. alpha-Carbon protonation and, in some cases, protonation of one of the carbonyl oxygens to form an enol compete with alkoxide or thiolate ion expulsion. This rendered the kinetic analysis more complex but allowed the determination of pK(a) values and of proton-transfer rate constants at the alpha-carbon. In conjunction with previously reported data on the nucleophilic addition of alkoxide and thiolate ions to the same Meldrum's acid derivatives, rate constants for nucleophilic addition by the respective neutral alcohols and thiols could also be calculated. Various structure-reactivity relationships are discussed that help define transition-state structures. Comparisons with similar reactions of alkoxide ion adducts of beta-alkoxy-alpha-nitrostilbenes provide additional insights.