Biomedical subjects

T T Tibbitts

Publications and source records attributed to T T Tibbitts.

6 recordsLinked to original sources

PubMed1996

Kinetic and structural consequences of replacing the aspartate bridge by asparagine in the catalytic metal triad of Escherichia coli alkaline phosphatase.

In each subunit of the homodimeric enzyme Escherichia coli alkaline phosphatase, two of the three metal cofactors Zn2+ and Mg2+, are bound by an aspartate side-chain at position 51. Using site-specific mutagenesis, Asp51 was mutated both to alanine and to asparagine to produce the D51A and D51N enzymes, respectively. Over the range of pH values examined, the D51A enzyme did not catalyze phosphate ester hydrolysis above non-enzymic levels and was not activated by the addition of millimolar excess Zn2+ or Mg2+. Replacement of Asp51 by asparagine, however, resulted in a mutant enzyme with reduced activity and a higher pH optimum, compared with the wild-type enzyme. At pH 8.0 the D51N enzyme showed about 1% of the activity of the wild-type enzyme, and as the pH was raised to 9.2, the activity of the D51N enzyme increased to about 10% of the value for the wild-type enzyme. Upon the addition of excess Mg2+ at pH 9.2, the D51N enzyme was activated in a time-dependent fashion to nearly the same level as the wild-type enzyme. The affinity for phosphate of the D51N enzyme decreased tenfold as the concentration of Mg2+ increased. Under optimal conditions, the k(cat)/K(m) ratio for the D51N enzyme indicated that it was 87% as efficient as the wild-type enzyme. To investigate the molecular basis for the observed kinetic differences, X-ray data were collected for the D51N enzyme to 2.3 angstroms resolution at pH 7.5, and then to 2.1 angstroms resolution at pH 9.2 with 20 mM MgCl2. The two structures were then refined. The low magnesium, low pH D51N structure showed that the third metal site was unoccupied, apparently blocked by the amide group of Asn51. At this pH the phosphate anion was bound via one oxygen atom, between the zinc cations at the first and second metal sites, which strongly resembled the arrangement previously determined for the D153H enzyme at pH 7.5. In the high magnesium, high pH D51N structure, the third metal site was also vacant, but the phosphate anion bound closer to the surface of the enzyme, coordinated to the first metal site alone. Electron density difference maps provide evidence that magnesium activates the D51N enzyme by replacing zinc at the second metal site.

Alkaline Phosphatase↗

PubMed1995

Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases.

In order to understand some of the differences between human placental, human, Saccharomyces cerevisiae and Escherichia coli alkaline phosphatases in specific activity, activation by magnesium, and pH versus activity profiles, the X-ray crystal structures of three mutant E. coli alkaline phosphatases have been determined. The aligned sequences of alkaline phosphatases from mammalian, yeast and E. coli show that 25 to 30% of the amino acids are absolutely conserved and the active site residues are completely conserved with the exception of residues 153, 328 and 155. The bacterial enzyme has a salt-bridge, Asp153/Lys328, near the third metal binding site which, based on sequence homology, is apparently absent in the yeast and mammalian enzymes. The human enzymes have histidine at positions 153 and 328, and the yeast enzyme has histidine at position 328. In the E. coli enzyme, Asp153 was replaced by histidine (D153H), Lys328 was replaced by histidine (K328H), and a double mutant (DM) was constructed containing both mutations. The structure of the K328H enzyme was refined using cross-validation to a resolution of 2.3 A with a working R-factor of 0.181 and a free R-factor of 0.249. The DM structure was determined to a resolution of 2.5 A with a working R-factor of 0.166 and a free R-factor of 0.233. The structure of the D135H enzyme, which has been reported to a resolution of 2.4 A, has been re-refined using cross-validation to a working R-factor of 0.179 and a free R-factor of 0.239 for controlled comparisons with the two new structures. In all three structures the most significant changes are related to the bound phosphate inhibitor and the identity of the metal ion in the third binding site. The changes in the position of the phosphate group and the alterations at the third metal binding site indicate the structural basis for the variations in the steady-state kinetic parameters previously reported for these enzymes.

Alkaline Phosphatase↗

PubMed1995

Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites.

The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1994

Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.

Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.

Alanine↗

PubMed1993

Deconvolution of disoriented fiber diffraction data using iterative convolution and local regression.

Computationally efficient procedures are described for the deconvolution of disoriented fiber diffraction data to the resolution limit of measurable intensity in the patterns. The methods can be applied to diffraction data from imperfectly parallel arrays of one-dimensionally periodic rods or two-dimensionally periodic sheets, randomly rotated about their unique axes, to derive a representation of the intensity distribution corresponding to perfectly parallel orientation. With use of angular convolution and local angular regression, a set of uniform cylindrically averaged squared structure factors are iteratively adjusted, subject to a minimum-wavelength constraint, until they produce a disoriented pattern that fits the observed diffraction data. The results from this deconvolution provide a measure of the properly scaled cylindrically averaged squared structure factors, which can be used with other structural information to construct a physically plausible trial model suitable for further refinement. Sample deconvolutions of simulated X-ray patterns from partially oriented gap junction membranes are presented and the results from point-model deconvolutions are compared to those from constrained deconvolutions that began with the transform of a physically plausible trial model.

Intercellular Junctions↗

PubMed1990

Diffraction diagnosis of protein folding in gap junction connexons.

To diagnose the regular polypeptide conformation in gap junction membranes, the x-ray intensities diffracted from oriented specimens have been separated into a modulated component due to the coherently ordered portion of the channel-forming pairs of connexon hexamers and a diffuse component due to the disordered parts. The spherically averaged ordered protein diffraction, in the resolution range 15-4 A, was compared with intensity curves calculated from the Fourier transforms of proteins representative of the major tertiary structural classes. From this comparison the alpha-helical content of the ordered portion of the connexon was estimated to be approximately 60%. Calculation of cylindrically averaged patterns for oriented distributions of alpha-helical and beta-sheet proteins demonstrated that the ratio of the modulated diffracted intensity near 5 A spacing on the meridian and 10 A spacing on the equator observed from the gap junctions can be accounted for by alpha-helical segments inclined relative to the connexon axis. Model dimers of connexonlike hexamers were constructed from alpha-helix bundle proteins to correlate features in the calculated diffraction patterns with the model parameters. On the basis of these correlations, the ordered gap junction diffraction data indicate that alpha-helical segments centered at 38 A from the midplane of the gap have a mean radial location approximately 24 A from the hexamer axis, and an axial projected length of approximately 35 A. Thus, these alpha-helical segments traverse the hydrocarbon core of the lipid bilayer, as expected for the four hydrophobic sequences of the connexin molecule.

Animals↗