Changes in Nicotinamide Metabolism by One Amino Acid Deficiency. (I) Threonine-, Tryptophan-, Aspartic Acid-, Lysine-, Leucine-, or Methionine-free Diet

Effects of a threonine-, tryptophan-, aspartic acid-, lysine-, leucine-, or methionine-free diet fed to rats on the metabolism of nicotinamide were investigated. The body weights of rats and food intakes were greatly decreased by feeding of the diet excluding any of the above essential amino acids compared to the control group, however, not by feeding of an aspartic acid-free diet. The sum of the urinary excretion of nicotinamide, N¹-methylnicotinamide (MNA), N¹ -methyl-2-pyridone-5-carboxamide (2-Py), and N¹ -methyl-4-pyridone-3-carboxamide (4-Py) changed roughly in proportion to food intake. In the groups fed with the threonine- and lysine-free diets, the urinary excretion of MNA greatly increased compared with the control group during the whole experimental period and in the groups fed with the leucine- and methionine-free diets, increased excretion of MNA was observed on day o–day 1. Whenever the increase in MNA excretion was observed, a decrease in 4-Py excretion was observed. This was attributed to the activity of 4-Py-forming MNA oxidase decreasing when rats were fed with the diet excluding one of the essential amino acid except for tryptophan. Therefore, the (2-Py +4-Py)/MNA excretion was greatly decreased by feeding of the diet excluding one of the essential amino acids except for the tryptophan-free diet. These results strengthened our hypothesis that the (2-Py +4-Py)/MNA excretion reflects the adequacy of amino acid nutrition.     

Inhibition of urease activity by hydroxamic acid derivatives of amino acids.

Hydroxamic acids have been reported to be potent and specific inhibitors of urease (EC 3.5.1.5) activity of plant and bacterial origin. The present investigation was performed on the inhibitory effect of hydroxamic acid derivatives of naturally occurring amino acids on the urease activity of the Jack Bean and the alimentary tracts of rats. Methionine-hydroxamic acid was the most powerful inhibitor (I50=3.9 X 10(-6) M) among nineteen alpha-aminoacyl hydroxamic acids. Phenylalanine-, serine-, alanine-, glycine-, histidine-, threonine-, leucine-, and arginine-hydroxamic acids followed, in order of decreasing inhibitory power. The inhibition proceeded with time at a comparable rate to fatty acyl hydroxamic acid inhibition. The I50 values of alpha-aminoacyl hydroxamic acids were found to be almost equal to those of the corresponding fatty acyl hydroxamic acids. This fact shows that the alpha-amino group did not affect inhibitory power. However, aspartic-beta-, lysine-, and glutamic-gamma-hydroxamic acids, in descending order, were much less inhibitory, probably due to the presence of a carboxyl or omega-amino group. Furthermore, the pH optimum of the inhibition shifted to lower pH in the presence of a carboxyl group, and to a higher pH in e presence of an amino group. The results suggest that the dissociation of an acidic or a basic group reduces the inhibitory power of hydroxamic acid. Hydroxamic acid inhibits urease activity with strict specificity, excpet for aspartic-beta-hydroxamic acid, which inhibited asparaginase competitively. Hydroxamic acid derivatives of amino acids inhibited not only the urease activity of the Jack Bean, but also that of the caecum and ileum parts of the rat intestine.     

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines

Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.     

Complementing amino acid substitutions within loop 6 of the alpha/beta-barrel active site influence the CO2/O2 specificity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Photosynthesis-deficient mutant 45-3B of the green alga Chlamydomonas reinhardtii contains a chloroplast mutation that causes valine-331 to be replaced by alanine within the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. This amino acid substitution occurs in loop 6 of the alpha/beta-barrel active site, three residues distant from catalytic lysine-334. The mutation reduces the specific activity of the enzyme and also reduces its CO2/O2 specificity factor by 42%, but the amount of holoenzyme is unaffected. In a previous study, an intragenic-suppressor mutation, named S40-9D, was selected that causes threonine-342 to be replaced by isoleucine, thereby increasing the CO2/O2 specificity of the mutant enzyme by 36%. To determine which other residues might be able to complement the original mutation, nine additional genetically independent revertants have now been analyzed. Another intragenic suppressor, represented by mutation S61-2J, causes glycine-344 to be replaced by serine. This change increases the CO2/O2 specificity of the mutant enzyme by 25%. Of the revertants recovered and analyzed, the mutant enzyme was improved only due to true reversion or by intragenic suppression mediated by substitutions at residues 342 or 344. Changes in the physical properties of the two pairs of complementing substitutions indicate that steric effects within loop 6 are responsible for the observed changes in the CO2/O2 specificity of the enzyme.     

The role of lysine-132 and arginine-136 in the receptor-binding domain of the K99 fibrillar subunit.

The gene encoding the K99 fibrillar adhesin of Escherichia coli has been modified by oligonucleotide-directed, site-specific, mutagenesis. The tryptophan-67, lysine-132, lysine-133 or arginine-136 were replaced by leucine, threonine, threonine and serine, respectively. The threonine-133 mutant fibrillae were indistinguishable from wild-type fibrillae. In contrast, replacement of lysine-132 or arginine-136 by threonine or serine, respectively, resulted in mutant fibrillae which had completely lost adhesive capacity, suggesting that the positive charges of these residues are essential for the interaction with the negatively charged sialic acid residue of the receptor molecules. After the replacement of tryptophan-67 with leucine neither fibrillae nor subunits were detectable, indicating that the mutant product is unstable and that tryptophan-67 has an essential structural role in the K99 subunit.     

Synthetic peptide substrates for the membrane tyrosine protein kinase stimulated by epidermal growth factor

The substrate specificity of the epidermal-growth-factor-stimulated tyrosine protein kinase of A431 cell membranes has been studied using a series of synthetic peptide analogs of the sequence around the phosphorylated tyrosine-419 of pp60src. The nine-residue peptide Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Thr-Ala was phosphorylated on tyrosine with an apparent Km of 0.4 mM and a V of 5.7 nmol X min-1 X mg-1. Synthetic peptide tyrosine phosphorylation was stimulated by epidermal growth factor but not by insulin or relaxin. Extension of the nine-residue peptide to include the basic residues, arginine-412, arginine-422 and lysine-423 led to an increased apparent Km. Substitution of glutamic-418 by leucine also increased the apparent Km. In the model peptide Ile-Xaa-Xaa-Ala-Ala-Tyr-Thr-Ala a lower apparent Km was obtained when Xaa was glutamic rather than aspartic acid. Poly(aspartic acid) and poly(glutamic acid) had only weak effects on peptide tyrosine phosphorylation. The results support the concept that acidic residues and not basic residues are important specificity determinants for the epidermal-growth-factor-stimulated tyrosine protein kinase.     

Thrombin-bound conformation of the C-terminal fragments of hirudin determined by transferred nuclear Overhauser effects

The interaction of the C-terminal fragments (residues 52-65 and 55-65) of the thrombin-specific inhibitor hirudin with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution. Thrombin induces specific line broadening of the proton resonances of residues Asp(55) to Gln(65) of the synthetic hirudin fragments H-Asn-Asp-Gly-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH and acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH. This demonstrates that residues 55-65 are the predominant binding site of hirudin fragments with thrombin. Hirudin fragments take on a well-defined structure when bound to thrombin as indicated by several long-range transferred NOEs between the backbone and side-chain protons of the peptides, but they are not structured when free in solution. Particularly, transferred NOEs exist between the alpha CH proton of Glu(61) and the NH proton of Leu(64) [d alpha N(i,i+3)], between the alpha CH proton of Glu(61) and the beta CH2 protons of Leu(64) [d alpha beta(i,i+3)], and between the alpha CH proton of Glu(62) and the gamma CH2 protons of Gln(65) [d alpha gamma(i,i+3)]. These NOEs are characteristic of an alpha-helical structure involving residues Glu(61) to Gln(65). There are also NOEs between the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Distance geometry calculations suggest that in the structure of the thrombin-bound hirudin peptides all the charged residues lie on the opposite side of a hydrophobic cluster formed by the nonpolar side chains of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64).     

The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197.

The amino-acid sequences of two diphtheria toxin-related, non-toxic proteins, CRM45 and CRM197 , were deduced from the complete sequence of their genes: tox 45 and tox 197. CRM45 lacks the last 149 C-terminal amino-acid residues, but is otherwise identical to diphtheria toxin: a single C----T transition introduces an "ochre" (TAA) termination signal in tox 45, after the codon for threonine-386. A single G----A transition was also found in tox 197, leading to the substitution of glycine-52, present in the wild-type toxin, with glutamic acid in CRM197 . This aminoacid change is responsible for the loss of the NAD:EF2 ADP-ribosyltransferase activity in CRM197 , due most probably to an alteration of the NAD+ binding site.     

Human gastric lipase. The N-terminal tetrapeptide is essential for lipid binding and lipase activity

Human gastric lipase subjected to limited tryptic proteolysis lost its ability to hydrolyze emulsified long-chain triacylglycerol. Activity against a water-soluble substrate was however retained, indicating that proteolysis did not affect the active site. Sequence analysis revealed that trypsin specifically cleaved the linkage between lysine-4 and leucine-5. This cleavage rendered the enzyme unable to bind to emulsified triacylglycerol particles, e.g. human milk fat globules. We suggest that the N-terminal tetrapeptide, in particular lysine-4, is essential for the binding of human gastric lipase to lipid/water interfaces, and hence, for its physiological function.     

A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes

The lactose permease is an integral membrane protein that cotransports H(+) and lactose into the bacterial cytoplasm. Previous work has shown that bulky substitutions at glycine 64, which is found on the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a substantial decrease in the maximal velocity of lactose uptake without significantly affecting the K(m) values (Jessen-Marshall, A. E., Parker, N. J., and Brooker, R. J. (1997) J. Bacteriol. 179, 2616-2622). In the current study, mutagenesis was conducted along the face of TMS-2 that contains glycine-64. Single amino acid substitutions that substantially changed side-chain volume at codons 52, 57, 59, 63, and 66 had little or no effect on transport activity, whereas substitutions at codons 49, 53, 56, and 60 were markedly defective and/or had lower levels of expression. According to helical wheel plots, Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64 form a continuous stripe along one face of TMS-2. Several of the TMS-2 mutants (S56Y, S56L, S56Q, Q60A, and Q60V) were used as parental strains to isolate mutants that restore transport activity. These mutations were either first-site mutations or second-site suppressors in TMS-1, TMS-2, TMS-7 or TMS-11. A kinetic analysis showed that the suppressors had a higher rate of lactose transport compared with the corresponding parental strains. Overall, the results of this study are consistent with the notion that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport. We hypothesize that TMS-2 slides across TMS-7 and TMS-11 when the lactose permease interconverts between the C1 and C2 conformations. This idea is discussed within the context of a revised model for the structure of the lactose permease.     

An E. coli RuvC mutant defective in cleavage of synthetic Holliday junctions.

Escherichia coli RuvC protein is a specific endonuclease that resolves recombination intermediates into viable products. The structural features needed for RuvC activity were investigated by sequencing three ruvC mutations and relating the base pair changes identified to the activity of the mutant proteins. Each of the three mutations is a single base-pair substitution. ruvC51 converts glycine-15 to an aspartic acid residue. The product of ruvC51 was purified and shown to retain the ability to bind junctions, albeit with a slightly reduced affinity. However, it has lost the ability to resolve these structures by symmetrical cleavage. A multicopy ruvC51 plasmid confers sensitivity to UV light in a ruvC+ strain. The ruvC53 allele causes a glycine-17 to serine substitution while ruvC55 produces a stop codon. Neither of these genes produces a stable product. The results suggest that the N-terminal domain of RuvC may be concerned with cleavage of junctions.     

Site-directed mutagenesis of catalytic active-site residues of Taka-amylase A.

The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis: aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no alpha-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.     

The specificity of purified porcine pancreatic elastase

An electrophoretically homogeneous elastase preparation free from tryptic and chymotryptic activities was obtained by chromatography on DEAE-Sephadex and CM-cellulose. This preparation exhibits a narrower specificity towards peptide bonds than that observed by Naughton & Sanger (1961). With oxidized insulin B chain as substrate, the fastest breaks occur between alanine-14 and leucine-15 and between valine-18 and cysteic acid-19. The bond between glycine-23 and phenylalanine-24 is also efficiently hydrolysed. Other bonds hydrolysed are that between valine-12 and glutamic acid-13 and that between serine-9 and histidine-10. Oxidized insulin A chain is hydrolysed only at one of two points, between alanine-8 and serine-9 or between serine-12 and leucine-13, and the rate of hydrolysis is very low.     

Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.

A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was found to be 4.3 microM at the optimum pH of 4.5. Reaction products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis or by 252Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22 degrees C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D.A700 increase of 0.100) produced at 37 degrees C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC50 of 27 nM when tested in a dose-response study with the purified enzyme. The apparent Ki for pepstatis was 2.9 nM. Several synthetic inhibitors of the enzymes were identified with IC50's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC50 of 1.3 nM and apparent Ki of 0.17 nM.     

Structural Basis for Non-Covalent Interaction Between Ubiquitin and the Ubiquitin Conjugating Enzyme Variant Human MMS2

Modification of proteins by post-translational covalent attachment of a single, or chain, of ubiquitin molecules serves as a signaling mechanism for a number of regulatory functions in eukaryotic cells. For example, proteins tagged with lysine-63 linked polyubiquitin chains are involved in error-free DNA repair. The catalysis of lysine-63 linked polyubiquitin chains involves the sequential activity of three enzymes (E1, E2, and E3) that ultimately transfer a ubiquitin thiolester intermediate to a protein target. The E2 responsible for catalysis of lysine-63 linked polyubiquitination is a protein heterodimer consisting of a canonical E2 known as Ubc13, and an E2-like protein, or ubiquitin conjugating enzyme variant (UEV), known as Mms2. We have determined the solution structure of the complex formed by human Mms2 and ubiquitin using high resolution, solution state nuclear magnetic resonance (NMR) spectroscopy. The structure of the Mms2–Ub complex provides important insights into the molecular basis underlying the catalysis of lysine-63 linked polyubiquitin chains.     

Enzymatic synthesis of chromogenic substrates for Glu,Asp-specific proteinases.

Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.     

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.     

Structure and function of Hb Saint-Jacques (alpha 2 beta 2 140 (H18) Ala----Thr): a new high-oxygen-affinity variant with altered bisphosphoglycerate binding

A low P50 value in a fresh red blood cell suspension was discovered in a polycythemic patient (Hb 19 g X dl-1). Routine acid and alkaline electrophoreses of the hemolysate were identical to normal hemolysate. Isoelectrofocusing (pH gradient 6-8) did not reveal any abnormal band whether performed with the fully liganded or deoxygenated samples. Precise analyses of the oxygen dissociation curves of the propositus' red cells demonstrated a biphasic Hill plot, a normal Bohr effect and low interaction with 2,3-bisphosphoglycerate (2,3-DPG). Studies on the unfractionated hemolysate confirmed these observations and the inhibition of the effect of organic phosphates. Structural studies were carried out on the mixture of beta A + beta X chains and revealed the presence of two beta Tp14 peptides. Sequencing the abnormal beta Tp14 peptide showed the substitution Ala----Thr of the beta 140 (H18) residue. This new variant was named Hb Saint-Jacques. Examination of the three dimensional model of HbAo indicates that the substitution beta 140 (H18) Ala----Thr induces van der Waals interactions with the nearby lysine-82 (EF6) and leucine-81 (EF5) and a displacement of the EF corner of the beta chains. This is likely to change the normal position of the lysine-82 (EF6), a major anionic binding site in the central cavity between the two beta chains. Functional studies confirm the interpretation of a steric hindrance inhibiting the binding of large organic phosphates to Hb Saint-Jacques.     

Variant forms of galanin isolated from porcine brain

In a peptide concentrate, prepared from acid extracts of porcine brain, several galanin-like immunoreactive peptides were detected and two of these were purified. Characterization of the peptides by sequence analysis, mass spectrometry, and capillary zone electrophoresis identified them as a N-terminally nine residue elongated form of galanin, preprogalanin(24–61) amide, and as an N-terminally four residue truncated form of galanin corresponding to preprogalanin(37–61) amide. The former finding suggests that the removal of the signal peptide in preprogalanin occurs by enzymatic cleavage between glycine-23 and leucine-24. The presence of truncated galanin might refer to a mechanism, where galanin is inactivated by removal of functionally important amino acid residues from the N-terminus.