A dimeric two-component receiver domain inhibits the sigma54-dependent ATPase in DctD.
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Adenosine Triphosphatases↗
Biomedical subjects
G K Farber
Publications and source records attributed to G K Farber.
Distributions of structural features contributing to thermostability in mesophilic and thermophilic alpha/beta barrel glycosyl hydrolases.
Analysis of the structural basis for thermostability in proteins has come mainly from pairwise comparisons of mesophilic and thermophilic structures and has often yielded conflicting results. Interpretation of these results would be enhanced by knowing the normal range of features found for mesophilic proteins. In order to provide the average and distribution values of structural features among similar mesophilic proteins, we compared the amino acid composition, solvent accessible surface area, hydrogen bonds, number of ion pairs, and thermal factors of 22 structures of alpha/beta barrel glycosyl hydrolases. These distributions are then compared to values from seven alpha/beta barrel glycosyl hydrolases from thermophilic organisms. We find that the distribution of each structural feature is broad within the mesophilic proteins and illustrates the difficulty of making pairwise comparisons of mesophiles to thermophiles where differences for individual proteins may be within the normal range for the group. In comparing mesophiles to thermophiles as a group, we find that thermophilic structures have fewer glycines in a particular region of the structure and higher thermal factors at room temperature. These results suggest the basis for thermostability may be related to protein motion rather than to static features of protein structure.
Amino Acids↗
Crystallographic analysis of solvent-trapped intermediates of chymotrypsin.
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Chymotrypsin↗
New approaches to rational drug design.
Rational drug design has emerged as a powerful technique. In this review, three new developments in rational drug design are explored. These developments include new methods to find binding sites for small molecules on the surface of a protein, the suggestion that the protein environment may change the shape of a protein sufficiently to alter drug design, and the use of data emerging from structural genomics in drug design. Although these are three new and distinct areas, the insights derived from these studies suggest a reason for the observation that similar drugs do not always bind to a protein in the same manner.
Binding Sites↗
Breaking the low barrier hydrogen bond in a serine protease.
The serine protease subtilisin BPN' is a useful catalyst for peptide synthesis when dissolved in high concentrations of a water-miscible organic co-solvent such as N,N-dimethylformamide (DMF). However, in 50% DMF, the k(cat) for amide hydrolysis is two orders of magnitude lower than in aqueous solution. Surprisingly, the k(cat) for ester hydrolysis is unchanged in 50% DMF. To explain this alteration in activity, the structure of subtilisin 8397+1 was determined in 20, 35, and 50% (v/v) DMF to 1.8 A resolution. In 50% DMF, the imidazole ring of His64, the central residue of the catalytic triad, has rotated approximately 180 degrees around the Cbeta-Cgamma bond. Two new water molecules in the active site stabilize the rotated conformation. This rotation places His64 in an unfavorable geometry to interact with the other members of the catalytic triad, Ser221 and Asp32. NMR experiments confirm that the characteristic resonance due to the low barrier hydrogen bond between the His64 and Asp32 is absent in 50% DMF. These experiments provide a clear structural basis for the change in activity of serine proteases in organic co-solvents.
Crystallography, X-Ray↗
Crystallization and preliminary X-ray studies of the Rhizobium meliloti DctD two-component receiver domain.
The Rhizobium meliloti DctD two-component receiver domain was expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using the hanging-drop vapor-diffusion geometry with ammonium phosphate as the precipitant. The crystals diffract to 2.3 A and exhibit the symmetry of space group I222 or I212121. The unit-cell dimensions are a = 59.0, b = 58.6 and c = 169.8 A. The asymmetric unit contains a dimer and the crystals have a Vm of 2.16 A3 Da-1.
Bacterial Proteins↗
Crystallization and preliminary X-ray studies of Pseudomonas putida histidine ammonium-lyase.
Histidine ammonium-lyase from P. putida was expressed in Escherichia coli, purified to homogeneity, and crystallized by the vapour-diffusion method using polyethylene glycol 3350 as the precipitant. The crystals, which diffract to at least 2.5 A resolution, exhibit the symmetry of space group P212121, with unit-cell parameters a = 89.7, b = 138.2 and c = 164.8 A. The asymmetric unit contains a tetramer, and the crystals have a Vm value of 2.41 A3 Da-1.
Bacterial Proteins↗
The structure of L-aspartate ammonia-lyase from Escherichia coli.
The X-ray crystal structure of l-aspartate ammonia-lyase has been determined to 2.8 A resolution. The enzyme contains three domains, and each domain is composed almost completely of alpha helices. The central domain is composed of five long helices. In the tetramer, these five helices form a 20-helix cluster. Such clusters have also been seen in delta-crystallin and in fumarase. The active site of aspartase has been located in a region that contains side chains from three different subunits. The structure of the apoenzyme has made it possible to identify some of the residues that are involved in binding the substrate. These residues have been examined by site-directed mutagenesis, and their putative roles have been assigned [Jayasekera, M. M. K., Shi, W., Farber, G. K., & Viola, R. E. (1997) Biochemistry 36, 9145-9150].
Aspartate Ammonia-Lyase↗
Evaluation of functionally important amino acids in L-aspartate ammonia-lyase from Escherichia coli.
The high-resolution structure of l-aspartate ammonia-lyase from Escherichia coli has recently been determined [Shi, W., Dunbar, J., Jayasekera, M. M. K., Viola, R. E., & Farber, G. K. (1997) Biochemistry 36, 9136-9144]. An examination of the putative active site has been carried out, with the active site located in a cleft that contains the functionally significant lysine 327. A list of potential active site residues has been generated based on their proximity to this active site lysine, sequence homology comparisons with other members of the aspartase-fumarase enzyme family, and the necessity for chemically reasonable functionalities for the proposed roles. The five most likely candidates in the putative active site cleft have been examined by site-directed mutagenesis to test their feasibility for either substrate binding or acid-base catalytic roles. Arginine and lysine residues have been identified that appear to function in the orientation and binding of aspartic acid at the enzyme active site. Some tentative assignments have also been made of the acid and base catalytic groups that are proposed to be involved in the deamination reaction.
Amino Acids↗
Laue crystallography: Lights! Camera! action!
Recent advances in the Laue method of X-ray data collection from protein crystals have allowed very short-lived reaction intermediates to be observed successfully.
Bacterial Proteins↗
The effect of denaturants on protein structure.
Virtually all studies of the protein-folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X-ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study.
Crystallography, X-Ray↗
Direct measurement of reactivity in the protein crystal by steady-state kinetic studies.
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Chymotrypsin↗
Laue crystallography. It's show time.
New crystallographic techniques make it possible to observe directly all of the intermediates in an enzymatic reaction. Such a series of structures can be combined to create a detailed movie of enzymatic catalysis.
Binding Sites↗
The evolution of sugar isomerases.
L-Arabinose isomerase (EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose. Here we report on the purification, kinetic mechanism and chemical mechanism of L-arabinose isomerase from Escherichia coli. The enzyme catalyzes the isomerization of L-arabinose to L-ribulose by a proton transfer mechanism, in contrast to xylose isomerase which uses a hydride transfer mechanism to perform a similar isomerization. Arabinose isomerase activity is metal dependent, although the enzyme can catalyze the exchange of the proton attached to carbon 2 of arabinose with the solvent in the absence of metal ion. Manganese(II) is the only metal ion which renders the enzyme active for the isomerization reaction. Arabinose isomerase has high substrate specificity for L-arabinose. The difference in chemical mechanism between xylose isomerase and arabinose isomerase suggests that these enzymes are not related by convergent evolution. This work also suggests that unless convergent evolution has been demonstrated, the mechanism of one enzyme may not give any insight into the mechanism of a second enzyme catalyzing the same reaction.
Aldose-Ketose Isomerases↗
The structure and evolution of alpha/beta barrel proteins.
Roughly 10% of all known enzyme structures have an alpha/beta barrel domain. The members of this large family of proteins catalyze very different types of reactions. Such diversity of function has made this family a target for protein engineering. The evolutionary history of this family has been the subject of vigorous debate. In this paper, arguments are made to support the divergence of all members of this family from a common ancestor. Because of the lack of strong sequence homology, the ancestral molecule must be very old. A hypothesis concerning the relationship between chemical mechanism and evolutionary history is discussed. Evidence is presented to suggest that convergent molecular evolution occurs when there is only one energetically reasonable pathway for a chemical reaction.
Amino Acid Sequence↗
X-ray crystal structure of gamma-chymotrypsin in hexane.
Crystals of gamma-chymotrypsin grown in aqueous solution were soaked in n-hexane, and the structures of both the soaked and the native crystals were determined to 2.2-A resolution. Seven hexane molecules and 130 water molecules were found in the hexane-soaked crystals. Two of the seven hexane molecules are found near the active site, and the rest are close to hydrophobic regions on or near the surface of the enzyme. In the hexane structure, water molecules that were not observed in the native structure form a clathrate around one of the hexane molecules. Only 97 water molecules were found in the native structure. The temperature factors for atoms in the hexane environment are lower than those in the aqueous environment. There are significant changes between the two structures in the side chains of both polar and neutral residues, particularly in the vicinity of the hexane molecules. These changes have perturbed the hydrogen-bonding patterns. The electron density for the peptide bound in the active site has been dramatically altered in hexane and appears to be tetrahedral at the carbon that is covalently bound to Ser 195. The crystalline enzyme retains its active conformation in the nonpolar medium and can catalyze both hydrolysis and synthesis reactions in hexane.
Amino Acid Sequence↗
Isotopic exchange plus substrate and inhibition kinetics of D-xylose isomerase do not support a proton-transfer mechanism.
The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+- or Mn(2+)-dependent enzyme that catalyzes the aldose-ketose isomerization of xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15, 25 and 55 degrees C shows < 0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of pH had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F NMR experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 mM 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating H2S release upon reaction of xylose isomerase with 1-thioglucose. D-Xylose isomerase-catalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 degrees C. No change in rate-determining step occurs over this temperature range. 13C NMR experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.
Aldose-Ketose Isomerases↗