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Protein and Peptide Letters, Vol. 5, No. 6, 1998

Contents

Review

The Promiscuous Active Site Specificity/Binding Preferences of the Fungal Aspartic Proteinase, Rhizopuspepsin. Pp. 303-316.
W. Todd Lowther and Ben M. Dunn
[Abstract]

Regular Papers

Effects of Reaction Intermediate Analogs on the Thermal Stability of Bovine Adenosine Deaminase. Pp. 317-320.
Edward M. Cook, Bijal S. Patel and B. Mark Britt
[Abstract]

310 Helix Formation in Protected Tripeptide. Pp. 321-332.
R. Jayakumar, M. Aulice Scibioh, Vasantha Pattabhi and P.T Manoharan
[Abstract]

Novel Applications of Purification Procedures to a-Gliadins. Pp. 333-340.
J.B. Turner, G.V. Garner, D.B. Gordon, G.A. Lord and C.A. Smith
[Abstract]

Multiple Attack of Action of a-Galactosidase from Trichoderma reesei. Pp. 341-348.
Konstantin A. Shabalin, Elena V. Eneyskaya, Anna A. Kulminskaya, Andrew N. Savel'ev and Kirill N. Neustroev
[Abstract]

Crystallisation Report

Crystallization and Preliminary X-ray Analysis of b-Amylase from Bacillus cereus var. mycoides. Pp. 349-354.
Takuji Oyama, Masami Kusunoki, Yoji Kishimoto, Yoshiyuki Takasaki and Yasunori Nitta
[Abstract]

Abstracts

[Back to top] The Promiscuous Active Site Specificity/Binding Preferences of the Fungal Aspartic Proteinase, Rhizopuspepsin. W. Todd Lowther and Ben M. Dunn.
Rhizopuspepsin, a secreted proteinase from the fungus Rhizopus chinensis, has been used as a model system for studying the active site interactions of the aspartic proteinases. Substrates containing systematic substitutions at P5-P1 and P2'-P3' were analyzed in an effort to identify molecular interactions that could be exploited in structure-based drug design. The recombinant enzyme showed a high degree of kinetic similarity to naturally occurring isozymes. Rhizopuspepsin was able to cleave substrates with side chains varying widely in size, shape, charge and hydrophobicity. Specificity constant (kcat/Km) values (0.06 to 3.84 s-1 mM-1) were consistently two to five-fold higher than for the same peptides studied with the mammalian enzymes, porcine pepsin, cathepsin D and cathepsin E. The structural basis for this broad specificity, particularly in the S2 subsite, was rationalized to be a result of glycine residues present at positions 290 and 292.

[Back to top] Effects of Reaction Intermediate Analogs on the Thermal Stability of Bovine Adenosine Deaminase. Edward M. Cook, Bijal S. Patel and B. Mark Britt.
The effects of inosine, the ground state analog 2-aminopurine riboside, and the transition state analog coformycin on the thermal stability of bovine adenosine dearninase were determined in order to assess the extent to which the different ligand types stabilize enzyme global structure. It is found that only coformycin substantially stabilizes the enzyme. We interpret this as evidence that binding of the reaction transition state is accompanied by a stabilizing enzyme global conformational change. Repercussions in terms of general enzyme catalysis are discussed.

[Back to top] 310 Helix Formation in Protected Tripeptide. R. Jayakumar, M. Aulice Scibioh, Vasantha Pattabhi and P.T Manoharan.
The conformational analysis of a synthetic peptide Boc-Lys(Z)-Gly-Val-NHMe has been carried out, as a model for nucleating segment in helix formation. 1H NMR studies (270 MHz) suggested that the Gly (2) NH, Val (3) NH and NHMe groups are solvent shielded. Conformational energy calculations and intramolecular hydrogen bonding constrains favour 310 helix structure for the peptide. Theoretical and spectroscopic results are consistent with the presence of a transannular 4 -> 1 hydrogen bond between Lys (1) CO and NHMe with Gly (2) NH and Val (3) being sterically shielded from the solvent environment.

[Back to top] Novel Applications of Purification Procedures to a-Gliadins. J.B. Turner, G.V. Garner, D.B. Gordon, G.A. Lord and C.A. Smith.
Novel procedures using repeated chromatographic separations are described for the exhaustive purifications of selected a-gliadins of wheat flour. The molecular masses of isolated pure samples were determined using matrix assisted laser desorption ionisation - time of flight - mass spectrometry (MALDI-TOF-MS) and are compared with values estimated from analyses of acid hydrolysates. The partial amino acid sequence obtained by automated Edman degradation of a highly purified a3-gliadin is presented and compared with published data.

[Back to top] Multiple Attack of Action of a-Galactosidase from Trichoderma reesei. Konstantin A. Shabalin, Elena V. Eneyskaya, Anna A. Kulminskaya, Andrew N. Savel'ev and Kirill N. Neustroev.
The presence of multiple attack of action of a-galactosidase from Trichodenna reesei was demonstrated in hydrolysis of p-nitrophenyl-6-O-a-galactopyranosyl-O-galactopyranoside using NMR, GLC-MS and absorption spectra in the visible region The degree of multiplicity of attack in this reaction appeared to be 2.1 and depended on pH. Studying some other glycosidases revealed the absence of the multiplicity in the hydrolysis of p-nitrophenyl oligosaccharides.

[Back to top] Crystallization and Preliminary X-ray Analysis of b-Amylase from Bacillus cereus var. mycoides. Takuji Oyama, Masami Kusunoki, Yoji Kishimoto, Yoshiyuki Takasaki and Yasunori Nitta.
Crystals of the b-amylase from Bacillus cereus var. mycoides were grown by the vapor diffusion method with polyethylene glycol 6000 as the precipitant. These crystals belong to the monoclinic system, space group C2 with a = 177.9 Å, b = 112.9 Å, c = 146.2 Å and b = 105.8°. They diffracted well to 2.0 Å resolution on a synchrotron radiation source, a full data set being collected to 2.2 Å resolution. On the basis of the Matthews' parameter (Vm) at least four molecules are estimated to be in the asymmetric unit.