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The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(l, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-[beta]NA (4-acetyloxyproline [beta]-naphthylamide) was a better substrate than Pro-[beta]NA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyrl50, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria.

The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular models found in the non-redundant protein sequence database. The information content of the amino acid sequence of a typical LA module is investigated by constructing sequences with alanine residues at nonconserved positions in the module.

The composite nature of the spider dragline silk with respect to its protein constituents is emphasized. It is found that at least two spidroins contribute to the formation of the thread, with Masp 1 seemingly representing the predominant protein while other spidroin-like proteins are likely to be added as a coat layer.

The goal of this study is to use the model system described earlier to make direct measurements of the enthalpy of helix formation at different temperatures. For this we studied model alanine peptides in which helix formation can be triggered by metal ([La.sup.3+]) binding. The heat of [La.sup.3+] interaction with the peptides at different temperatures is measured by isothermal titration calorimetry. Circular dichroism spectroscopy is used to follow helix formation. Peptides of increasing length (12-, 16-, and 19-aa residues) that contain a L[a.sup.3+]-binding loop followed by helices of increasing length, are used to separate the heat of metal binding from the enthalpy of helix formation. We demonstrate that (i) the enthalpy of helix formation is - 0.9 [+ or -] 0.1 kcal/mol; (ii) the enthalpy of helix formation is independent of the peptide length; (iii) the enthalpy of helix formation does not depend significantly on temperature in the range from 5 to 45[degrees] C, suggesting that the heat capacity change on helix formation is very small. Thus, the use of metal binding to induce helix formation has an enormous potential for measuring various thermodynamic properties of [alpha]-helices.

A study investigated isotropic and anisotropic hyperfine coupling constants of selected nuclei in some alanine-derived radicals. It is believed that this investigation represents the first, comprehensive quantum chemical study of the electron paramagnetic resonance spectroscopic properties of alanine. Calculated hyperfine interaction values were compared with available experimental data. Experimental methods, findings and conclusions are discussed.

The internal electron transfer reactions and proton-transfer coupled electron transfer without dioxygen in mutant enzymes, where I-362 and I-359 were substituted by KM(I-362) and TA(I-359), respectively, were examined in cytochrome c oxidase obtained from Rhodobacter sphaeroides. The results demonstrated that the electron transfer involving hemes a3 and a, having a time constant of approximately three microseconds, were not influenced in the mutant enzymes. On the other hand, the electron transfer between hemes a3 and a was reduced in the TA(I359) and KM(I-362) variants.

The influence of alpha-amonoisobutyric acid (Aib) on the structural integrity of a globular protein was examined using a model system of C-terminal fragment 255-316 of thermolysin. The results demonstrated that the rational inclusion of a globular protein of Aib may result in substantial stabilization of the protein structure. The Aib replacement in the polypeptide chain may have great implications for engineering protein stability, provided that an appropriate method for assimilating Aib into a protein is discovered.

Phosphinothricin-tripeptide synthetases from Strectomyces viridochromogenes were obtained and characterized to elucidate the enzymology Pt tripeptide (Pt tripeptide) synthesis. The S. viridochromogenes was found to contain N-acetyl-demethylphosphinothricin- (AcDMPt) and alanine-activating enzymes and bind their substrates as thioesters. The results provided evidence that AcDMPt is the direct antecedent in the assembly of Ptt, with 62-kilodalton enzymes from Ptt-generating S. viridochromogenes reacting with AcDMPt but not Pt as thioester and adenylate.

The characterization of the dadAB operon from Klebsiella aerogenes strains is conducted in an experiment to illustrate how strains that failed to catabolize alanine were able to form a glutamate auxotrophy. In this study, the K. aerogenes strain is grown in minimal medium that contains glucose, ammonium and alanine. The dad operon is mediated by a nitrogen assimilation control protein and activated by the Ntr system.

The presence of three putative metal binding amino acid residues and a catalytic-based residue were identified through the use of a mutagenesis strategy applied to the histidine, glutamate and aspartate positions of dipeptidase VanX. It was indicated that his116, asp123 and his184 were loci for zinc-dipeptidase interaction residues. The zinc coordination was found to be homologous to the N-terminal domain of Streptomyces albus G zinc-dependent carboxypeptidase.

Relative energetics of several alanine tetrapeptide conformations were determined. Results have shown that the large errors in hydration free energies in a given series in continuum-dielectric calculations could be explained by the inaccuracy of the potential functions in reproducing quantum chemical pair hydrogen bonding energies. It was indicted that the force fields that use fixed atom-centered charges will not reproduce energetic trends.

An unstable complex that converts to (cyclotetra-L-alanyl)oxotechnetium(V) anion is produced from the complexation of technetium with tetra-L-alanine. The assumption is that in the initial complex, an oxotechnetium(V) core is coordinated to three amide nitrogen atoms and the amine nitrogen atom of tetra-L-alanine. Amide formation and cyclization is facilitated since the carboxyl group is forced in the vicinity of the amine group. Cyclization of tetra-L-alanine takes place at no carrier added and carrier added scales.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 ([Delta]2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 ([Delta]2-27), and 2B4 ([Delta]2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6-to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenyl-ethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added [H.sub.2][O.sub.2], but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

The amino acid at position 45 in ferredoxin of the cyanobacterium Anabaena influences the spectroscopic properties exhibited by the protein. Replacement of alanine by serine changes the spectroscopic, redox and electron transfer properties of the protein. Alanine occupies the residue at position 45 in the cyanobacterial protein, while serine is present in higher plant ferredoxins at the same position. The position is close to the iron atom that is involved in redox reactions.

Stark-modulated free-expansion jet spectrometer helps identify two conformers of beta-alanine and assign the rotational spectrum of beta-alanine. Intramolecular interactions in these conformers of beta-alanine are similar to those in both gaseous glycine and alpha-alanine. Ab initio molecular calculations predict an energetically favorable third conformer, but experimental studies do not support the presence of a third conformer.

A study of the Na-dependent alanine transport features in membrane vesicles from thin and heavy Zucker rat skeletal muscles and the effect of insulin treatment on the transport systems reveals that both lean and obese rats have comparable kinetic constants for the alanine uptake that varies with Na. While the alanine transport in lean rats is enhanced by 1.8 times after activation by insulin, obese rat membranes do not exhibit any variations in the transport. The variation in alanine transport accounts for the abnormal metabolism of muscle proteins in animals.

Reverse transcription and polymerase chain reaction help clone and sequence the entire cDNA for topoisomerase II alpha from human melanoma cell lines FEM-X and Fvp3 and this cloning helps investigate the mechanism of VP-16 resistance in FEM-X cells. Examination of the clone reveals a 3 bp deletion in Fvp3, leading to the deletion of Ala 429. Genetic studies show that Ala 429 deletion from topoisomerase II alpha suppresses the susceptibility of the enzyme to drug-induced cleavage.

Double-stranded DNA templates induce alpha-helical conformation in peptides that comprise alanine and lysine. These templates are associated with basic-helix-loop-helix and basic-leucine zipper regulatory proteins which bind to symmetric DNA sites as dimers.

The production of D-glutamic acid is catalyzed by glutamate racemase activity in Escherichia coli. The D-glutamic acid is required for bacterial cell-wall peptidoglycan production. The enzyme is purified to homogeneity, and the kinetic properties are determined by the use of murI gene product of Escherichia coli.

The uptake and efflux of alanine, lysine and proline in the cytoplasmic membranes of Acinetobacter species is executed by single high-affinity carriers for each solute. Lysine and alanine are transported by a membrane potential and a transmembrane pH gradent. A membrane potential and the transmembrane Na+ gradient act as the driving force for proline transport. Both H+ and Na+ function as coupling ions in the amino acid translocation in the polyphosphate-accumulating A. johnsonii strain.

The mutant zinc finger peptide PAPA analysis based on ADR1 C-terminal region indicates the presence of alanine residues in place of wild-type proline residues. The residue after second cysteine ligand is at a positive phi angle common to all zinc finger peptides. Aspartic and arginine acid bonds are observed in the peptide, in the absence of DNA similar to Zif268 protein. DNA binding sites and ADR1b and PAPA in Zif268 protein are studied to determine the zinc finger domain mutants that affect the DNA binding sites.

The amino acid Phe112, which covalently bonded to allosteric inhibitor of dCMP deaminase (dTTP) in T4-phage dCMP deaminase, is present in a peptide pattern, originally conserved in various dTTP binding proteins. Site-directed mutagenesis is used to study enzyme response changes to allosteric effectors when Phe112 was converted to an alanine.

A study of residue side chains reveals that the tissue factor extracellular domain possibly adopts the cytokine receptor homology unit folding pattern with two seven-beta-strand modules. Mutational analysis suggests that important residues are spatially closer and present as a cluster on the beta-strand module boundary. Residues involved in ligand binding occur in positions corresponding to ligand interactive residues.

Studies of the design of unnatural polypeptide backbones that fold in compact and specific patterns reveal that intra-molecular hydrogen bonds between neighboring amide groups in the polypeptide partly controls the folding pattern. Studies of beta-alanine and gamma-amino butyric acid reveal that the former favors hydrogen bonding between neighboring amides. Variable temperature FT-IR studies yield thermodynamic data for hydrogen bond-controlled peptide folding.

Proteolysis regulation in rat hepatocyte suspensions is evaluated using leucine as the regulatory amino acid and alanine as the coregulator. Macroautophagy and its control by amino acids occur simultaneously in liver cell populations. The multiphase's control by amino acids is an inherent characteristic of the hepatocyte, which can be used to study the putative plasma membrane site of leucine identification.

Pre-steady state kinetic analysis along with site-directed mutagenesis helps examine the function of threonine 234 in catalysis of tyrosyl adenylate formation by tryosyl-tRNA synthetase from Bacillus stearothermophilus. The transition state is stabilized by threonine 234 during the development of tyrosyl adenylate in tyrosyl-tRNA synthetase. Double mutant free energy cycles facilitate the examination of the function of coupling between T234 and K230.

Helix-forming exceptions are seen in structured peptides, which include natural sequences such as S-peptide from ribonuclease A1 and P-alpha 5 from BTPI-2 and other designed polyalamine-based peptides having regular Lys and Glu insertions. Molecular dynamics and Monte Carlo simulations of capped undecaalanine peptide are conducted to determine helical conformation in water. Molecular dynamics simulation starting at log 3 rapidly converts into an alpha-helix, which is stable by ca. 1.0 kilocalorie per mole per residue.

The reaction of Citrobacter freundii tyrosine phenol-lyase with L- as well as d-alanine were investigated. The results reveal the discovery of separate quinoid intermediates and the initial assessment of the total rate constants for alanine racemization in which tyrosine was the catalyst. These data give a basis for future experiments on the alpha,beta-excluding interaction.

Alanine replaces proline to reveal the maintenance of the wild-type structure with the cis-peptidyl association at 1.7 angstroms resolution, where the crystal geometry of a human carbonic anhydrase II (CAII) variant, cis-proline-202, transforms to alanine (P202A). The wild type, CO2 hydrase activity and attraction for sulfonamide hindering factors of P2O2A CAII resemble one another. The fall of the constancy of the folded state, due to the replacement of cis-proline by cis-alanine, is associated with the denaturation, stimulated by hydrochloride. This reveals the occurrence of the unfolded state and an equilbrium intermediate in guanidine.

D-alanine was examined to discover if it was caused by bacteria by making ddY/DAO mice devoid of germs, then gnotobiotic and finally measuring the alanine in their urine. A greater amount of D-alanine was found in the urine of mutant ddY/DAO- mice that did not have D-amino-acid oxidase activity compared to the normal ddY/DAO+ mice and the D-alanine from intestinal bacteria. The metabolism of D-amino acids obtained from bacteria was a physiological activity of the enzyme.

The mantle musculature and blood samples from the unstrained and cannulated squid under controlled conditions after exhaustion from high levels of exercise are studied to determine the concentrations of intermediate- and end-products of anaerobic energy metabolism and of the free amino acid content. The energy production before fatigue is contributed by the anaerobic metabolism, for which proline is the substrate. The hemocyanin function in the species Loligo and Illex is protected by the acidification of protons in the tissue.

Catheterized, depancreatized (PX) and normal (C) dogs were studied to estimate insulin-deficient gluconeogenesis regulation. Hepatic alanine uptake, arterial levels, hepatic concentrations and net gut output were unaltered during rest in C and PX. During exercise, all factors increased in PX, with only a fall in alanine concentration in C. Lactate induced gluconeogenesis was lesser in C than in PX at rest due to the enhanced efficiency of intrahepatic gluconeogenesis. Hepatic load increase and larger output of gut lactate and alanine also accounted for enhanced gluconeogenesis in the diabetic state.

D-alanine, along with D- and L-lactic acid, may be useful as molecular markers for bacterial contamination. Heat treatment and storage do not alter the D-alanine content in juices. D-alanine levels greater than 1 ppm or D/D+L percentage greater than 2 may indicate bacterial contamination in industrial juices.

Steroid target tissues regualte the amount of ligand that is available to bind and transactivate steroid receptors. The principles that govern steroid hormone binding and steroid turnover by enzymes are analyzed using rat 3Alpha-hydroxysteroid dehydrogenase as a protein model.

The secondary structure of tetraalanine in water is determined by combining polarized Raman, FTIR, vibrational circular dichroism(VCD) and electronic circular dichroism(ECD). Tetraalanine exhibits a higher propensity for PPII than trialanine for which a 50:50 mixture of polyproline II and an extend beta-strand-like conformation was obtained from recent spectrosopic studies.

Conformational preferences of the alanine dipeptide in water are studied by a variety of experimental techniques. The issue of what interactions are responsible for the predominance of P11 is studied explicitly without relying on simplified models.

The role of alanine sequences in the formation of beta sheets of fibroin-like protein, MaSp 1, of Nephila clavipes was determined through FT-IR spectroscopy. Results have shown that the beta sheets were lined up by amino acid sequences which consisted of stretches of four and seven alanines sandwiched by GAG and GGAG termini. It was indicated that the peptides did not exhibit aggregation at high concentrations.

Chelative complexes, formed by alpha-amino acids with an organic boronic acid, are characterized by a complexation which is responsible for an elevation in liquid membrane transport by a phenylboronic acid/trioctylmethylammonium bromide (PBA-TOMA) admixture. The formation of alpha-amino acids are classified as hydrophilic species and require intricate polar balance by binding-site residue to be rendered mobile by being transferred from bulk water to binding proteins.

An analysis of a nonapeptide Boc-Val-deltaPhe-Phe-Ala-Phe-deltaPhe-Val-Phe-Gly-OCH3 bearing three deltaPhe residues provided its crystal structure. Design of polypeptide helical residues can include deltaPhe as conformation constraining residues. One-to-one concurrence between the numbers of deltaPhe residues and that of beta-bands is absent.

Computer simulations were used in observing the conformational equilibria in the poly-alanine peptides, (Ala(sub 8)(super +)), (Ala(sub 16)(super +)) and the lysine-doped poly-alanine peptide (Ac-(AAAAK)(sub 3)A-NH(sub 2)(super 3+)). Two-dimensional umbrella sampling techniques along with multiple time step molecular dynamics were used in carrying out the computer simulations. Results indicated that the 3K peptide has a helical structure in vacuum and in solution. The poly-alanine peptides form compact complexes in vacuum so that solvation will occur on the N-terminus.