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J M Manning

Publications and source records attributed to J M Manning.

At least 55 records · Page 3Linked to original sources
PubMed1995

A recombinant human hemoglobin with asparagine-102(beta) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride.

A recombinant (r) mutant hemoglobin (Hb) with Asn-102(beta) replaced by an Ala (N102A(beta)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast. The side chain of Asn-102(beta) is part of an important region of the alpha 1 beta 2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn-102(beta) and Asp-94(alpha) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation. This proposal has been tested by expression of an rHb containing alanine at position 102(beta); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface. The nature of the desired replacement was established by sequencing the entire mutated beta-globin gene as well as the tryptic peptide containing the substitution. Further characterization by SDS-PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb. Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1995

Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.

Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, beta-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.

Alanine↗
PubMed1995

Role of leucine 201 of thermostable D-amino acid aminotransferase from a thermophile, Bacillus sp. YM-1.

We studied the catalytic role of leucine 201 residue of the thermostable D-amino acid aminotransferase: the residue was shown crystallographically to be in the vicinity of the active site to interact with the bound pyridoxal phosphate. We replaced the leucine 201 by alanyl or tryptophanyl residues by means of site-directed mutagenesis. The L201A and L201W mutant enzymes showed anomalous kinetic behavior in the overall reaction. The reaction rates of the L201A and L201W mutant enzymes gradually decreased with an increase in the reaction time to become practically zero at a high concentration of substrates. The mutant enzymes were also inactivated in the half reaction with D-alanine, although more slowly than in the overall reaction. The absorption spectra of the mutant enzymes in the presence of D-alanine and alpha-ketoglutarate suggest that the enzyme molecules were mostly in the pyridoxamine form under the conditions employed. These phenomena were explained by assuming two (or more) enzyme species showing kinetically different catalysis for pyridoxamine form of the mutant enzymes, and the rate of conversion from one of these pyridoxamine forms to the pyridoxal form should be very low. The leucine 201 residue probably regulates the function of cofactor during the reaction of D-amino acid aminotransferase.

Alanine↗
PubMed1994

Interaction of gamma-glutamyl transpeptidase with acivicin.

Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine. Reactivation (more than 85% complete) is associated with release from the inactivated enzyme of compounds that exhibit the properties of threo-beta-hydroxy-L-gamma-glutamyl hydroxamate and 3-hydroxypyrrolidone-2-carboxylate. We found that the enzyme acts very slowly on acivicin, at a rate that is about 10(-9) that of its normal catalytic rate with glutathione, to form threo-beta-hydroxy-L-glutamate and hydroxylamine. The findings indicate that inhibition by acivicin involves its transformation on the enzyme to an inhibitory species which is attached, apparently by ester linkage, to a specific hydroxyl group of the enzyme. The very slow rate of release of this intermediate appears to account for the observed inhibition.

Animals↗
PubMed1994

Human acylpeptide hydrolase. Studies on its thiol groups and mechanism of action.

The presence of a cysteine residue(s) near the active site of acylpeptide hydrolase was suggested by inactivation of the enzyme with sulfhydryl-modifying agents and by the substantial protection against inactivation afforded by the competitive inhibitor acetylmethionine. 5,5'-dithiobis-(2-nitrobenzoate) titrations of the native and the denatured enzyme together with analysis for cysteic acid after performic acid oxidation showed that the enzyme contained 12 free SH groups and three disulfide bonds/monomer. Chemical modification with radiolabeled iodoacetamide led to the labeling of Cys-30 and Cys-64 suggesting that one or both of these Cys residues are close to the active site. Modification of one or both of them probably inhibits the enzyme either because of a distortion of the active site or because the adducts present a barrier to the efficient diffusion of substrates into and products out of the active site. Studies on the mechanism of action of acylpeptide hydrolase have employed p-nitrophenyl-N-propyl carbamate as a potent active site-directed inhibitor. Enzyme inactivation, which follows pseudo first-order kinetics, is diminished by the competitive inhibitor acetylmethionine. The inhibited enzyme slowly regains activity at a rate that is increased in the presence of the nucleophile hydroxylamine. A general mechanism involving an acyl-enzyme intermediate is supported by evidence for the formation of acetyl-alanyl hydroxamate during hydrolysis of acetyl-alanine p-nitroanilide in the presence of hydroxylamine. The effect on Vmax and Km during this reaction indicate that hydrolysis of the acyl-enzyme intermediate is rate-limiting.

Amino Acid Sequence↗
PubMed1994

Properties of a recombinant human hemoglobin double mutant: sickle hemoglobin with Leu-88(beta) at the primary aggregation site substituted by Ala.

A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.

Alanine↗
PubMed1994

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.

Amino Acid Sequence↗
PubMed1994

Determination of free D-amino acids with a bacterial transaminase: their depletion leads to inhibition of bacterial growth.

A general procedure is described to determine the common free D-amino acids except D-proline in mixtures that also contain L-amino acids. The system employs exogenous pure bacterial D-amino acid transaminase coupled with 2-oxohexanoate, which accepts the amino group from D-amino acids to form D-norleucine. This amino acid is readily quantified by amino acid analysis since it elutes in a position not occupied by any of the common amino acids. Formation of norleucine denotes the presence of some D-amino acid(s) whose identity can be established by a corresponding decrease in the susceptible amino acid(s) after treatment. The utility of the procedure is demonstrated by determination of the amounts of free D-alanine and free D-glutamate in extracts of Escherichia coli JM-103 grown on minimal medium; D-alanine was the major D-amino acid. By the same principle, 2-oxohexanoate through coupling with endogenous bacterial D-amino acid transaminase is shown to be capable of inhibiting the growth of E. coli by depleting it of the D-alanine and D-glutamate.

Amino Acids↗
PubMed1994

Dietary polyunsaturated fat decreases interaction between low density lipoproteins and arterial proteoglycans.

Polyunsaturated dietary fat (n-3 and n-6) results in less atherosclerosis in monkeys compared to lard (Parks, J.S., Kaduck-Sawyer, J., Bullock, B.C., and Rudel, L.L., Arteriosclerosis 10, 1102-1112; Rudel, L.L., Parks, J.S., Johnson, F.L., and Babiak, J., J. Lipid Res. 27, 465-474, 1986). We hypothesized that this was due, in part, to a decreased reactivity of low density lipoproteins (LDL) with arterial proteoglycans (PG). To test this hypothesis, cynomolgus monkeys were fed diets containing lard, safflower oil (n-6 polyunsaturated; Poly), menhanden fish oil (FO), or oleic acid-rich safflower oil (oleinate; Mono) for 14 mon, and plasma LDL were isolated and characterized. Several properties of LDL thought to be important in the interaction of LDL with arterial PG were measured including LDL particle size, chemical composition, sialic acid content, density distribution, apolipoprotein E (apoE) content and cholesteryl ester transition temperature. Plasma LDL cholesterol concentrations (mg/dL) after 14 mon of diet consumption averaged (mean +/- SEM): FO (366 +/- 45), Lard (352 +/- 27), Poly (279 +/- 24), and Mono (230 +/- 43). The composition of LDL was similar among diet groups except that FO LDL were relatively depleted of cholesteryl ester and enriched in protein and were smaller in size. LDL sialic acid content was similar among diet groups (4.5-5.0 micrograms/mg LDL protein). The LDL apoE/B molar ratio, a measure of the apoE content per LDL particle averaged: Mono (3.0 +/- 1.0), Poly (2.0 +/- 0.1), Lard (1.8 +/- 0.5), and FO (1.0 +/- 0.2).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Random chemical modification of hemoglobin to identify chloride binding sites in the central dyad axis: their role in control of oxygen affinity.

Knowledge of the mechanism by which chloride and carbon dioxide lower the oxygen affinity of hemoglobin may aid in the design of new blood substitutes since these allosteric regulators permit hemoglobin (Hb) to release its O2. Stable covalent modifiers of hemoglobin, used either in a selective or a random mode, have been used to elucidate the binding sites of CO2 or chloride. For determination of CO2 binding, specific chemical modification of Hb by the carboxymethylation reaction was used. To identify the oxygen-linked chloride binding sites, random chemical modification of Hb was employed.

Binding Sites↗
PubMed1993

Biochemical and functional properties of recombinant human sickle hemoglobin expressed in yeast.

Previous studies had indicated that recombinant and natural human sickle hemoglobin had similar chemical properties (Martin de Llano, J. J., Schneewind, O., Stetler, G., and Manning, J. M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 918-922). In the present study, additional biochemical and physiological characterization of some primary through quaternary structural features of recombinant sickle hemoglobin are described. The molecular weight of the purified recombinant sickle hemoglobin was identical to natural sickle hemoglobin as determined by mass spectrometry, thus excluding extensive post-translational modification in the yeast system. Carboxypeptidases A and B together catalyzed the release of COOH-terminal amino acids at the same rate for recombinant and natural hemoglobin S, consistent with identity in their primary and secondary structures in this region of the molecule. The tryptic peptide maps of natural and recombinant hemoglobins were practically indistinguishable, indicating the same internal protein sequences for recombinant and natural hemoglobins. As a probe of the secondary structure of recombinant sickle Hb, the reactivity of the SH group of Cys-93(beta) was investigated for the glutathione sickle hemoglobin adduct, which has significant anti-gelling and anti-sickling properties. The position of glutathione at Cys-93(beta) was established by direct mass spectrometric analysis of enzyme digests; reduction of this derivative to the unmodified chains was also observed by mass spectrometry and by isoelectric focusing. The oxygen equilibrium curves of recombinant and natural sickle hemoglobin at high protein concentration were superimposable with identical Hill coefficients of 3.3. The response of recombinant sickle hemoglobin to chloride with respect to a lowered oxygen affinity was identical to that of natural sickle hemoglobin. The gelation properties of recombinant and natural sickle hemoglobins were identical at the high hemoglobin concentrations that occur in the red cell. Therefore, the yeast expression system synthesizes a completely functional recombinant sickle hemoglobin with the same biochemical and physiological properties as natural sickle hemoglobin with respect to features characteristic of its primary through quaternary structures.

Amino Acids↗
PubMed1993

Crystallization and preliminary X-ray studies of human erythrocyte acylpeptide hydrolase.

Crystals of acylpeptide hydrolase suitable for structure determination have been obtained. This enzyme removes the N-terminal formyl or acetyl group together with the first amino acid residue from N-terminal blocked peptides including bioactive peptides. One set of crystals, which diffract to 2.2 A, are in space group P2 with cell dimensions a = 118.6 A, b = 82.3 A, c = 182.1 A, beta = 91.6 degrees. The search for suitable heavy-atom derivatives is underway.

Amino Acid Sequence↗
PubMed1993

Role reversal for substrates and inhibitors. Slow inactivation of D-amino acid transaminase by its normal substrates and protection by inhibitors.

D-Amino acid transaminase, which catalyzes the synthesis of D-alanine and D-glutamate for the bacterial cell wall, is a candidate for the design of specific inhibitors that could be novel antimicrobial agents. Under the experimental conditions usually employed for enzyme assays, kinetic parameters for its substrates were determined for short incubation periods, when intermediates and products do not accumulate and the enzyme activity is linear with time. Such kinetic analyses indicate that the enzyme accepts most D-amino acids but D-aspartate and D-glutamate are the best substrates. Under a different type of experimental conditions when the enzyme is exposed to D-alanine, intermediates, and products for periods of hours, it slowly becomes inactivated (Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., and Manning, J. M. (1992) Biochemistry 31, 6018-6023). We now report that D-aspartate, D-glutamate, and L-alanine also lead to slow inactivation. Methylation or amidation of the alpha-COOH group of D-alanine prevents inactivation, indicating that decarboxylation is required for inactivation; the slow release of CO2 from substrate is demonstrated. The alpha-methyl analog of D-alanine, D-aspartate, and D-glutamate do not lead to inactivation, showing that the alpha-hydrogen of the substrate is required, i.e. that some processing is required. Lys145, which binds pyridoxal 5'-phosphate in the wild-type enzyme, is not involved in the inactivation since two active site mutant enzymes, K145Q and K145N, are also inactivated. Reactivation of the inactive enzyme at acidic pH is accompanied by the release of ammonia corresponding to 1 mol/mol of dimeric enzyme. Competitive inhibitors, amine-containing buffers, and thiols effectively impede the inactivation. This reversal in the roles of substrates and inhibitors, i.e. when a substrate can be an inactivator and an inhibitor can act as a protector, occurs during a time period not usually used to measure steady-state kinetics or initial velocities of enzyme reactions and could have physiological relevance in cells.

Amino Acids↗
PubMed1993

Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase.

D-Amino acid transaminase (EC 2.6.1.21), from Bacillus sp. YM-1, a thermostable enzyme with pyridoxal 5'-phosphate as coenzyme and a target for the design of novel antimicrobial agents, catalyzes the reversible transfer of an amino group between D-alanine and alpha-ketoglutarate to form pyruvate and D-glutamate, respectively. To explore the catalytic role of Lys-145, which binds the coenzyme, a site-specific mutant enzyme, K145Q (in which Lys-145 had been mutated to glutamine) constructed earlier (Futaki, S., Ueno, H., Martinez del Pozo, A., Pospischil, M. A., Manning, J. M., Ringe, D., Stoddard, B., Tanizawa, K., Yoshimura, T., and Soda, K. (1990) J. Biol. Chem. 265, 22306-22312) was compared to the wild-type enzyme for its kinetic parameters. Initial velocity studies and partial reaction isotope exchange experiments showed that the low activity of the mutant enzyme (about 1.5% the activity of the wild-type enzyme with saturating substrates) is an intrinsic property, confirming that contaminating enzymes do not account for the low activity of the K145Q mutant enzyme. The rates of the forward reaction for both wild-type and mutant enzymes were 30-40 times higher than the rates of the reverse reaction. KM values for the four substrates were 10 to 100 higher for the mutant compared to the wild-type enzyme. Whereas D-alanine is preferred over L-alanine by the wild-type enzyme (10(3) higher kcat/KM for D- over L-alanine), the K145Q enzyme does not efficiently discriminate between L- and D-alanine. Both wild-type and mutant enzymes also catalyze the slow racemization of L- and D-alanine. Proton NMR studies showed that wild-type enzyme catalyzed a time-dependent exchange of the C alpha proton of D-alanine with solvent D2O and a slow exchange of the alpha proton of L-alanine; the latter slow exchange rate is the same for the C alpha proton of both L- and D-alanine with the K145Q mutant enzyme. Thus, in addition to binding pyridoxal 5'-phosphate, the active-site Lys-145 of D-amino acid transaminase is involved in several other important functions, i.e. it optimizes catalytic efficiency and it maintains stereochemical fidelity. The steady-state kinetic results on the K145Q mutant enzyme together with the findings on the relative racemization rates and the NMR protein exchange data suggest that an alternate base catalyzes abstraction of the alpha proton of substrate in this mutant D-amino acid transaminase.

Bacillus↗
PubMed1993

Recombinant human sickle hemoglobin expressed in yeast.

Sickle hemoglobin has been expressed in the yeast Saccharomyces cerevisiae after site-directed mutagenesis of a plasmid containing normal human alpha- and beta-globin genes. Cassette mutagenesis of this plasmid was achieved by inserting a DNA fragment containing the beta-globin gene in the replicative form of M13mp18 to make a point mutation and then reconstituting the original plasmid containing the mutated beta-globin gene. Pure recombinant hemoglobin S was shown to be identical to natural sickle hemoglobin in its ultraviolet and visible absorption bands and by gel electrophoresis, isoelectric focusing, amino acid analysis, mass spectrometry, partial N-terminal sequencing, and functional properties (P50, cooperativity, and response to 2,3-bisphosphoglycerate). In yeast and in mammalian cells, cotranslational processing yields the same N-terminal valine residues of hemoglobin alpha- and beta-chains, but in bacterial expression systems the N terminus is extended by an additional amino acid because the initiator methionine residue is retained. Since the N-terminal valine residues of both chains of hemoglobin S participate in important physiological functions, such as oxygen affinity, interaction with anions, and the Bohr coefficient, the yeast expression system is preferable to the bacterial system for recombinant DNA studies. Hence, mutagenesis employing this expression system should permit definitive assignments of the role of any amino acid side chain in hemoglobin S aggregation and could suggest additional approaches to therapeutic intervention. The engineering of this system for the synthesis of sickle hemoglobin and its purification to homogeneity in a single column procedure are described.

Amino Acid Sequence↗