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D L Birdsall

Publications and source records attributed to D L Birdsall.

10 recordsLinked to original sources
PubMed1998

Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase.

We have determined structures of binary and ternary complexes of five Asn229 variants of thymidylate synthase (TS) and related their structures to the kinetic constants measured previously. Asn229 forms two hydrogen bonds to the pyrimidine ring of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). These hydrogen bonds constrain the orientation of dUMP in binary complexes with dUMP, and in ternary complexes with dUMP and the TS cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. In N229 mutants, where these hydrogen bonds cannot be made, dUMP binds in a misoriented or more disordered fashion. Most N229 mutants exhibit no activity for the dehalogenation of 5-bromo-dUMP, which requires correct orientation of dUMP against Cys198. Since bound dUMP forms the binding surface against which the pterin ring of cofactor binds, misorientation of dUMP results in higher Km values for cofactor. At the same time, binding of the cofactor aids in ordering and positioning dUMP for catalysis. Hydrophobic mutants, such as N229I, favor an arrangement of solvent molecules and side-chains around the ligands similar to that in a proposed transition state for ternary complex formation in wild-type TS, and kcat values are similar to the wild-type value. Smaller, more hydrophilic mutants favor arrangements of the solvent and side-chains surrounding the ligands that do not resemble the proposed transition state. These changes correspond to decreases in kcat of up to 2000-fold, with only modest increases in Km or Kd. These results are consistent with the proposal that the hydrogen-bonding network between water, dUMP and side-chains in the active-site cavity contributes to catalysis in TS. Asn229 has the unique ability to maintain this critical network, without sterically interfering with dUMP binding.

Asparagine↗
PubMed1998

The separate effects of E60Q in Lactobacillus casei thymidylate synthase delineate between mechanisms for formation of intermediates in catalysis.

X-Ray crystal structures of Lactobacillus casei thymidylate synthase (TS) mutant complexes of E60D with dUMP, and E60Q with dUMP or FdUMP, as well as ternary complexes with folate analog inhibitor CB3717, are described. The structures we report address the decrease in rate of formation of ternary complexes in the E60 mutants. Structures of ternary complexes of L.casei TS mimic ligand-bound TS just prior to covalent bond formation between ligands and protein. Ternary complex structures of L.casei TS E60Q show the ligands are not optimally aligned for making the necessary covalent bonds. Since CB3717 is an analog of the open, activated form of the cofactor, these structures suggest that the slow rate of ternary complex formation in E60 mutants is at least partly the result of impaired alignment of ligands in the active site after binding and activation of the cofactor. Binary complexes of TS E60Q and TS E60D with substrate (dUMP) show no change in dUMP position or occupancy. These results are consistent with the fact that Kd(dUMP) and Km(dUMP) are almost the same, and the rates of folate-independent debromination of 5-bromo-dUMP are even higher than for wild type TS.

Amino Acid Substitution↗
PubMed1996

Partitioning roles of side chains in affinity, orientation, and catalysis with structures for mutant complexes: asparagine-229 in thymidylate synthase.

Thymidylate synthase (TS) methylates only dUMP, not dCMP. The crystal structure of TS.dCMP shows sCMP 4-NH2 excluded from the space between Asn-229 and His-199 by the hydrogen bonding and steric properties and Asn-229. Consequently, 6-C of dCMP is over 4 A from the active site sulfhydryl. The Asn-229 side chain is prevented from flipping 180 degrees to and orientation the could hydrogen bond to dCMP by a hydrogen bond network between conserved residues. Thus, the specific binding of dUMP by TS results from occlusion of competing substrates by steric and electronic effects of residues in the active site cavity. When Asn-229 is replaced by a cysteine, the Cys-229 S gamma rotates out of the active site, and the mutant enzyme binds both dCMP and dUMP tightly but does not methylate dCMP. Thus simply admitting dCMP into the dUMP binding site of TS is not sufficient for methylation of dCMP. Structures of nucleotide complexes of TS N229D provide a reasonable explanation for the preferential methylation of dCMP instead of dUMP by this mutant. In TS N229D.dCMP, Asp-229 forms hydrogen bonds to 3-N and 40NH2 of dCMP. Neither the Asp-229 carboxyl moiety nor ordered water appears to hydrogen bond to 4-O of dUMP. Hydrogen bonds to 4-O (or 4-NH2) have been proposed to stabilize reaction intermediates. If their absence in TS N229D.dUMP persists in the ternary complex, it could explain the 10(4)-fold decrease in kcat/Km for dUMP.

Asparagine↗
PubMed1996

Entropy in bi-substrate enzymes: proposed role of an alternate site in chaperoning substrate into, and products out of, thymidylate synthase.

Three steps along the pathway of binding, orientation of substrates and release of products are revealed by X-ray crystallographic structures of ternary complexes of the wild-type Lactobacillus casei thymidylate synthase enzyme. Each complex was formed by diffusion of either the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate or the folate analog 10-propargyl-5,8-dideazafolate into binary co-crystals of thymidylate synthase with 2'-deoxyuridine-5'-monophosphate. A two-substrate/enzyme complex is formed where the substrates remain unaltered. The imidazolidine ring is unopened and the pterin of the 5,10-methylene-5,6,7,8-tetrahydrofolate cofactor binds at an unproductive "alternate" site. We propose that the presence of the pterin at this site may represent an initial interaction with the enzyme that precedes all catalytic events. The structure of the 2'-deoxyuridine-5'-monophosphate and 10-propargyl-5,8-dideazafolate folate analog complex identifies both ligands in orientations favorable for the initiation of catalysis and resembles the productive complex. A product complex where the ligands have been converted into products of the thymidylate synthase reaction within the crystal, 2'-deoxythymidine-5'-monophosphate and 7,8-dihydrofolate, shows how ligands are situated within the enzyme after catalysis and on the way to product release.

Binding Sites↗
PubMed1992

Crystal structure disposition of thymidylic acid tetramer in complex with ribonuclease A.

The crystal structure of ribonuclease A with bound thymidylic acid tetramer is reported at 2.5-A resolution. The diffusion of the tetramer into native orthorhombic crystals of the ribonuclease allows for the formation of a structurally stable complex where the single-stranded nucleic acid enters and leaves the enzyme's catalytic region in a persistent 5'-3' direction. The binding of the tetramer to the enzyme's surface is facilitated and mediated by electrostatic interactions between basic protein residues and nucleotide phosphates. Two pyrimidine nucleotides are bound to the enzyme's active site in a manner similar to that observed for other complexes between ribonuclease A and nucleic acid oligomers.

Animals↗
PubMed1984

Structure of a pleiomeric form of poly d(AT):poly d(AT).

A chemically simple polynucleotide duplex, poly d(AT):poly d(AT), has been trapped in a fibrous form with a complex helical secondary structure with a large (7.4 nm) axial repeat 24 nucleotides long. The motif which is repeated by the symmetry elements is a hexanucleotide in which two residues (both TpA) have the less common gauche minus conformation at C3'-O3' and consequently distinctive phosphate orientations. This reinforces earlier conclusions that PypPu nucleotides tend to have different shapes from PupPy nucleotides and that DNA surfaces may signal what base sequences lie beneath them. The morphological differences between this pleiomeric DNA polymer and closely-related, but more symmetrical allomorphs are just as great as those observed in short DNA fragments in crystals.

Base Sequence↗
PubMed1983

Wrinkled DNA.

The B form of poly d(GC):poly d(GC) in orthorhombic microcrystallites in oriented fibers has a secondary structure in which a dinucleotide is the repeated motif rather than a mononucleotide as in standard, smooth B DNA. One set of nucleotides (probably GpC) has the same conformations as the smooth form but the alternate (CpG) nucleotides have a different conformation at C3'-O3'. This leads to a distinctive change in the orientation of the phosphate groups. Similar perturbations can be detected in other poly d(PuPy):poly d(PuPy) DNAs such as poly d(IC):poly d(IC) and poly d(AT):poly d(AT) in their D forms which have tetragonal crystal environments. This suggests that such perturbations are intrinsic to all stretches of duplex DNA where purines and pyrimidines alternate and may play a role in the detection and exploitation of such sequences by regulatory proteins.

DNA↗
PubMed1980

Left-handed DNA helices.

The synthetic DNA polymerpoly d(GC) . poly d(GC) has been studied by X-ray diffraction of orientated fibres in which it has either the well known (A or B) randed conformations have been observed with poly d(AC) . poly d(GT) and with poly d(As4T) . poly d(As4T), demonstrating that the novel structure is accessible to any DNA segment with an alternating purine-pyrimidine base sequence.

Base Sequence↗