Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.     

Effects of sulfhydryl reagents on the binding and release of penicillin G by D-alanine carboxypeptidase IA of Escherichia coli.

Purified D-alanine carboxypeptidase IA of Escherichia coli is inhibited by penicillin G and binds penicillin G reversibly. The binding of penicillin to the enzyme is relatively insensitive to sulfhydryl reagents, while release of penicillin from the enzyme is severely inhibited by these reagents. The inhibition of release parallels the inhibition of carboxypeptidase activity by the sulfhydryl reagents. In the presence of the sulfhydryl reagent p-chloromercuribenzoate, an acyl-enzyme intermediate, produced by the reaction of carboxypeptidase IA with diacetyl-L-lysyl-D-alanyl-D-alanine, accumulates and can be isolated. These results indicate that binding of penicillin to carboxypeptidase IA occurs by an acylation step of the carboxypeptidase reaction, while penicillin release occurs by a deacylation step of the reaction. Only the latter is inhibited by sulfhydryl reagents.     

Mutational evidence for identity of penicillin-binding protein 5 in Escherichia coli with the major D-alanine carboxypeptidase IA activity.

The defect in D-alanine carboxypeptidase IA activity in the dacA11191 mutant of Escherichia coli was correlated with a defect in the release of penicillin G from penicillin-binding protein 5. The results suggest that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity of the wild type and that the mutation results in a defect in the deacylation step catalyzed by this enzyme.     

Hydroxylaminolysis of penicillin binding componenets is enzymatically catalyzed.

The hydroxylaminolysis of the penicilloyl moiety from [14C]penicillin G binding component (PBC) complexes of the Bacillus subtilis D-alanine carboxypeptidase and of the mixture of PBC's of Staphylococcus aureus was inhibited by denaturation of the complexes by heat (55 degrees), detergent (1% sodium dodecyl sulfate), or trichloroacetic acid. The kinetics of inhibition by denaturation were comparable to those of the inhibition of [14C]penicillin G binding to the PBC's and of carboxypeptidase activity of the B. subtilis enzyme under identical denaturing conditions. These data establish that the hydroxylaminolysis is an enzymatically catalyzed process suggesting that penicillin G is bound to an enzymatically active site. Treatment of the denatured [14C]penicillin G-carboxypeptidase complex with sodium borohydride or at pH 12 resulted in the release of the penicilloyl moiety. These results are consistent with a carboxylic ester bond for the penicilloyl-PBC instead of a thiolester linkage as was initially presumed.     

Crystallographic data for the DD-carboxypeptidase-endopeptidase of low penicillin sensitivity excreted by Streptomyces albus g.

The dd-carboxypeptidase-endopeptidase of low penicillin sensitivity that is excreted by Streptomyces albus G has been crystallized from a polyethylene glycol (M_r 6000 to 7500) solution at pH 8.0. X-ray examination of the prismatic crystals shows that the space group is P2₁ with unit cell dimensions $\text{a = 51.1}\text{A}\text{?}\text{, b = 49.7}\text{A}\text{?}\text{, c = 38.7}\text{A}\text{?}\text{, β = 100.6 °}$ and one molecule in the asymmetric unit. A crystal suspension made in 50 mm-Tris · HCl buffer (pH 8.0) supplemented with 5 mm-MgCl₂ and 16% ($\text{w}\text{v}$) polyethylene glycol exhibits enzyme activity on the substrate Ac₂-l-Lys-d-Ala-d-Ala.     

D-alanine carboxypeptidase activity of Micrococcus lysodeikticus released into the protoplasting medium.

Conversion of whole cells of Micrococcus lysodeikticus to protoplasts allowed the release of a soluble form of a D-alanine carboxypeptidase into the protoplasting medium. The enzyme cleaves the terminal D-alanine from the radioactively labelled UDP-N-acetylmuramyl-pentapeptide containing L-lysine as the diamino acid. However, the enzyme is only minimally active in this fraction so that it had to be enriched and partially purified before its properties could be studied. Chromatography on carboxymethyl-Sephadex removed the lysozyme used in the protoplasting of the cells. The material which was unadsorbed to the column was applied to an affinity chromatography column of Ampicillin-Sepharose. Most of the contaminating protein was washed from the column while the D-alanine carboxypeptidase adhered to the resin and could be eluted with 0.5 M Tris-HCl buffer pH 8.6. Some of the properties of the enzymic activity were studied using this preparation. The enzyme was activated by Mg2+ ions with a broad optimum from 15--35 mM. It was maximally active when NaCl at a concentrations of 0.06--0.08 M was added to the assay, and the pH curve was biphasic with an alkaline optimum. The Km for substrate was found to be 0.118 mM. Enzymic activity was completely inhibited by low concentrations of Ampicillin and penicillin G.     

Noncholecystokinin peptides in human serum which cause gallbladder contraction

In fasting human serum, cholecystokinin (CCK) is not the principal substance which causes $\text{in}$$\text{vitro}$ rabbit gallbldder contraction. Removal of CCK by affinity chromatography from fasting sera from 8 healthy adults reduced bioactivity only by 18 ± 4% (SEM). Unlike CCK, the bioactivity of serum was enhanced by 30 to 57% rather than destroyed by pronase and chymotrypsin respectively and was not inhibited by dibutyryl cGMP. Reduction of serum bioactivity by carboxypeptidase Y indicated that the bioactive substances in serum are peptides. On Sephadex G-50, bioactive substances eluted in positions different from any known form of CCK. Thus, the principal substances in fasting human serum causing $\text{in}$$\text{vitro}$ gallbladder contraction are not CCK but are most likely small peptides which act at receptors different from the receptors for CCK.     

A peptide-like inhibitor of N-methyltransferase in rabbit brain

After pretreatment with pheniprazine, rabbits were administered C-14-tryptamine i.v. and the lung was assayed for the N-methylated derivatives. Unoxidized tryptamine was present, but no N-methyl or N, N-dimethyltryptamine was found in this tissue, which contains high levels of N-methyltransferase. It appears that the indolamine-N-methyltransfer reaction is inhibited in the intact tissue. Our investigation of the possible inhibitory mechanism has led to the purification and characterization of a dialysable factor which inhibits the enzyme $\text{in}$$\text{vitro}$. The factor, which is present in most tissues, was purified from newborn rabbit brain. It is present in two forms, one having approximate mol. wt. 1,500 and one mol. wt. 1,300. Both were inactivated by crystalline trypsin. The 1,300 form was digested by carboxypeptidase A to a smaller, but still active form. It is suggested that these peptides may control $\text{in}$$\text{vivo}$ the activity of the non-specific N-methyltransferase against tryptamine and serotonin.     

Evidence for a difference in mechanism between the reactions with carboxypeptidase A of the (-) and (+) - enantiomers of a thiolester substrate.

Cinnamoyl-carboxypeptidase A_γ has been isolated from the reaction of the enzyme with (+)-$\text{S-(trans}$-cinnamoyl)-α-mercapto-β-phenylpropionate. This is the first instance in which an acyl-enzyme has been detected directly during the esterase action of carboxypeptidase A. Also, the failure to detect such a species in the carboxypeptidase A-catalyzed hydrolysis of the (?)-enantiomer of the thiolester under similar conditions provides the first experimental demonstration that an enzyme can act on different enantiomers of a substrate through different mechanisms.     

Viral nucleocapsid melting to measure protein: RNA interactions

Thermal denaturation of nucleocapsids of wild type (WT) vesicular stomatitis virus (VSV), containing only the nucleocapsid protein (N) and viral RNA, caused a “melting” that resulted in an A_260nm absorbance increase of 140%. The nucleocapsids of two temperature-sensitive ($\text{ts}$) VSV mutants, $\text{ts}$ G31BP and $\text{ts}$ G22, both underwent larger absorbance increases of 251% and 177% respectively, suggesting these nucleocapsids are complexed by weaker N protein: RNA interactions than the WT-VSV. Two other mutants, $\text{ts}$ G31 and $\text{ts}$ G41 underwent A_260nm increases either similar to, or smaller than, that measured with WT-VSV nucleocapsids. RNA synthesis by $\text{ts}$ G31BP in infected cells was also found to be decreased at elevated temperatures. This temperature sensitive defect in viral RNA metabolism in $\text{ts}$ G31BP may be the result of weaker protein:RNA interactions associated with the nucleocapsid.     

Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.

All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.     

Glycine activation of PEP carboxylase from monocotyledoneous C4 plants

Phosphoenolpyruvate carboxylase from $\text{Zea}\text{mays}$ leaves was found to be activated by L-glycine and inhibited by maleic acid, but was not affected by the effectors for the bacterial enzymes. The activating effect of L-glycine was observed with all the enzymes from leaves of several monocotyledoneous C₄ plants, while the enzymes from dicotyledoneous C₄ plants and mono- and dicotyledoneous C₃ plants were not activated by L-glycine. Maleic acid inhibited the enzyme activities of all the higher plants tested.     

Solubilization and characterization of the partially purified penicillin sensitive D-alanine carboxypeptidase of Neisseria gonorrhoeae.

Treatment of crude gonococcal cell envelopes with a solution of 2 M KCl + 1% Brij 36T resulted in the solubilization of a portion of the D-alanine carboxypeptidase activity of Neisseria gonorrhoeae envelopes. This soluble enzyme preparation was partially resolved by chromatography on a column of DEAE-cellulose. The partially purified enzyme eluted from the column with a gradient of NaCl (0-1 M), catalysed the release of D-alanine from a radioactively labelled UDP-N-acetylmuramyl-pentapeptide with a pH optimum of 8.6. The Km for the soluble enzyme acting on this substrate was 0.18 mM. The enzyme activity was sensitive to inhibition by low concentrations of the beta-lactam antibiotics, penicillin G, ampicillin, oxacillin and mecillinam.     

A new antibiotic with known resistance factors,G418, inhibits plant cells

Growth rates of two lines of tobacco ($\text{Nicotiana}\text{tabacum}$) cell suspension cultures were measured in the presence or absence of G418, a new 2-deoxystreptamine antibiotic related to Gentamycin. Cell growth rates of $\text{N}$. $\text{tabacum}$ cv. Burley were inhibited at drug concentrations as low as 1.65 × 10^?7 M. At 4 × 10^?7 M, the doubling time was increased from 1.5 days (control) to 2.3 days (treatment). The drug was lethal to cells at 4 × 10^?6 M, and inhibition was irreversible. Cells of $\text{N}$. $\text{tabacum}$ cv. Wisconsin 38 also were inhibited by the drug, although at slightly higher concentrations (ca. 2–5 fold).In view of our findings, G418 and its associated resistance factors could be of great value in plant genetic engineering.     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.     

Analytical studies on crotamine hydrochloride.

The neurotoxin crotamine, which is a basic low molecular weight protein, was isolated, in the form of its hydrochloride, from a South Brazilian rattlesnake (Crotalus durissus crotaminicus, Crotalus durissus terrificus) venom.Disc electrophoresis, carried out in 7% acrylamide by the method of Reisfeld, at pH 4.5, showed a single band for the purified toxin.The toxin showed the following amino acid composition: Asx₂, Ser₃, Glx₂, Pro₃, Gly₅, Cys₅, Met₁, Ileu₁, Leu₁, Tyr₁, Phe₂, Trp₂, Lys₉, His₂, and Arg₂, which corresponds to a minimum molecular weight of 4760. This assignment of minimum molecular weight is supported by the recovery of 1.0 mole of N-terminal $\text{Tyr}\text{5800}\text{g}$ of crotamine by the Sanger dinitrophenol (DNP) method, 1.0 mole of $\text{Tyr}\text{4880}\text{g}$ by the Udenfriend method, and by uv analysis, which gave a value of 4820. The odd number of half-cystine residues ($\text{5}\text{4760}\text{g}$) cannot be explained on the basis of available analytical data.Tyrosine was the only amino-terminal residue detectable by the Sanger DNP method. Glycine was identified as the only carboxyl-terminal residue by the hydrazinolysis method of Akabori and by release upon treatment with yeast carboxypeptidase.The H⁺ electrometric and Cl^? complexometric titrations of crotamine hydrochloride showed that about 13 moles of HCl are bound per 4760 g of the free base.     

Comparative studies on human carboxypeptidases B and N

A series of dicarboxylic acid bi-product analogs of lysine and arginine have been tested as competitive inhibitors of human pancreatic carboxypeptidase B and human plasma carboxypeptidase N. The most effective derivative was guanidinoethylmercaptosuccinic acid with K_is of 0.5 and 1.0 × 10^?6m for Carboxypeptidases B and N, respectively. Values for the all-carbon guanidinopropylsuccinic acid were similar. In addition the kinetic parameters, K_m and $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, have been determined for the hydrolysis of benzoyl-alanyl-lysine and benzoylalanyl-arginine by human Carboxypeptidases B and N. These substrates have been proposed for use in improved spectrophotometric assays. An enhanced affinity of these substrates versus benzoyl-glycyl-lysine or benzoyl-glycyl-arginine indicates a significant participation of the penultimate amino acid in catalysis of substrate.     

Modulators of monoamine oxidase in plasma

Addition of small amounts of plasma activated the deamination of tryptamine by platelet monoamine oxidase (MAO). At higher concentrations, plasma inhibited the deamination instead. The inhibition was increased with increasing amounts of plasma added. The inhibition was uncompetitive in nature, partially reversed by prior ultrafiltration of the plasma through PM30 membranes and completely reversed by protein precipitation of plasma with perchloric acid. Addition of high amounts of plasma $\text{in}$$\text{vitro}$ also inhibited the activity of bovine striatal MAO. The inhibition of striatal deamination of tryptamine by plasma was noncompetitive in nature, completely reversed by ultrafiltration through PM30 membranes and partially reversed by perchloric acid treatment. The inhibition of striatal deamination of serotonin was noncompetitive in nature, not reversed by ultrafiltration but completely reversed by perchloric acid treatment. The pattern of inhibition of platelet or striatal MAO by plasma was different from that induced by addition of bovine serum albumin (BSA). Low concentrations of BSA added $\text{in}$$\text{vitro}$ activated the deamination of tryptamine or serotonin by platelet or striatal MAO by decreasing the K_m, while higher concentrations also decreased the V_max. The presence of protein, non-albumin circulating modulators of platelet or striatal MAO in plasma is discussed.     

The amino acid sequence of toxin V from Anemonia sulcata

Preparations of the β-galactoside-binding lectin of bovine heart have been shown to stimulate $\text{in vitro}$ the sialylation of the oligosaccharide Ga1β1→4G1cNAc and asialo-α₁-acid glycoprotein by bovine colostrum β-D-galactoside α2→6 sialyltransferase. Kinetic data revealed that in the presence of lectin the K_m values for Ga1β1→4G1cNAc and CMP-NeuAc were reduced from 25.0 to 11.6 mM and from 0.42 to 0.19 mM respectively, but the K_m for asialo-α₁-acid glycoprotein and the V_max values for all three substrates were little affected. Stimulation by the lectin was partially inhibited by Fucα1→2Ga1β1→4G1cNAc. This, together with the effects of certain plant lectins, suggests that the stimulation of sialytransferase may be mediated through the carbohydrate-binding properties of the lectin.