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F C Neuhaus

Publications and source records attributed to F C Neuhaus.

At least 55 records · Page 3Linked to original sources
PubMed1974

On the mechanism of action of vancomycin: inhibition of peptidoglycan synthesis in Gaffkya homari.

Vancomycin inhibits the synthesis of peptidoglycan in membrane preparations from Gaffkya homari with uridine diphosphate-N-acetylmuramyl (UDP-Mur-NAc)-pentapeptide as substrate, but not with either UDP-MurNAc-tetrapeptide or UDP-MurNAc-tripeptide. These results are correlated with the specificity studies described by Perkins and Nieto for complex formation between the antibiotic and the peptide subunit. It is concluded that the formation of a complex between vancomycin and a postulated cell wall acceptor or between vancomycin and the enzymes involved in peptidoglycan synthesis does not contribute to the inhibitory action of this antibiotic. The mechanism of vancomycin action on peptidoglycan synthesis is clearly different from that of moenomycin and bacitracin. In the presence of these antibiotics, peptidoglycan synthesis is inhibited with both UDP-MurNAc-pentapeptide and -tetrapeptide as substrates. In addition, these results provide additional insight into the mechanism of phospho-MurNAc-pentapeptide translocase. For example, enhancement of the transfer of phospho-MurNAc-peptide from UDP-MurNAc-peptide to undecaprenyl-phosphate at low concentrations of vancomycin is observed with UDP-MurNAc-pentapeptide and not with -tetrapeptide. Complexation of vancomycin with undecaprenyl-diphosphate-MurNAc-pentapeptide, resulting in an ineffective intermediate, would increase the rate of transfer by preventing the reassociation of undecaprenyl-diphosphate-MurNAc-pentapeptide with the enzyme.

Carbon Radioisotopes↗
PubMed1974

Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide.

The incorporation of N-acetylmuramyl (MurNAc)-peptides from nucleotide-activated precursors (reference: uridine diphosphate [UDP]MurNAc-Ala(1)-dGlu(2)-Lys(3)- dAla(4)-dAla(5)) with incomplete or modified peptide subunits into peptidoglycan was studied with membrane preparations from Gaffkya homari. The effectiveness of their utilization at low and high concentrations was compared on the basis of the values of V(max)/K(m) and V(max), respectively. At low concentration, replacement of alanine by glycine in position 5 has a small effect on the activity of the peptidoglycan synthesizing system, whereas it has a significantly larger effect in positions 1 and 4. The importance of d-alanine in position 4 at low substrate concentrations is also observed with the incomplete UDP-MurNAc-peptides. For UDP-MurNAc-tripeptide and -tetrapeptide, V(max)/K(m) is 0.06 and 0.55, respectively, of the value for the -pentapeptide. At high substrate concentration, replacement of d-alanine by glycine in either position 1 or 5 decreases the activity to 0.37 of the value for the reference nucleotide, whereas replacement in position 4 has a smaller effect (0.74). The profiles established from V(max) and V(max)/K(m) with UDP-MurNAc-tripeptide, -tetrapeptide, and -pentapeptide show good correlation. At low concentration the specificity profiles of phospho-MurNAc-pentapeptide translocase, catalyzing the initial membrane reaction, are similar to those for the peptidoglycan synthesizing system; at high concentration, however, the profiles differ. The translocase appears to provide a primary specificity barrier at high substrate concentration for UDP-MurNAc-Ala-dGlu-Lys-dAla-dAla and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla, and at low concentration for UDP-MurNAc-Ala-dGlu-Lys and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla. Moreover, it is suggested that an additional specificity barrier exists in the peptidoglycan synthesizing system for certain nucleotides. Thus, the cytoplasmic enzymes and the membrane-associated enzyme(s) cooperate to insure the formation of functioning peptidoglycan in this organism.

Adenosine Triphosphate↗
PubMed1974

Incorporation of D-alanine into the membrane of Streptococcus pyogenes and its stabilized L-form.

A principal aim of this study was to explain our earlier finding of a lack of d-alanine in the glycerol teichoic acid from the membrane of a stabilized L-form of Streptococcus pyogenes (B. M. Slabyj and C. Panos, 1973. J. Bacteriol. 114:934-942). It was found that the incorporation of d-alanine into the membrane teichoic acid of S. pyogenes requires either supernatant fraction or two enzymes from supernatant fraction, stimulator (d-alanine activating enzyme) and d-alanine:membrane acceptor ligase, plus membrane fragments, ATP and Mg(2+). A similar system from the L-form is inoperative. Also, no incorporation is observed with L-form or coccal supernatant fractions when L-form membranes are used. However, d-alanine incorporation is observed when L-form enzymes are used with membrane fragments from the parental streptococcus. Thus, the L-form possesses the required soluble components for d-alanine incorporation but the L-form membrane cannot function as acceptor even though it contains d-alanine-deficient membrane teichoic acid. These results suggest that a defect has occurred in the membrane of this stabilized L-form for d-alanine incorporation into membrane teichoic acid.

Adenosine Triphosphate↗
PubMed1972

Factors affecting the level of alanine racemase in Escherichia coli.

Alanine racemase occupies a key position in the alanine branch of peptidoglycan biosynthesis. The level of this enzyme in Escherichia coli W is a function of the carbon source. For example, growth on l-alanine causes a 25-fold higher level of alanine racemase when compared with growth on glucose. When potential inducers of this enzyme are added to either a glucose or succinate medium, a low specificity is observed with those compounds that cause higher levels of enzyme. Growth of E. coli W on either pyruvate, d-alanine, or l-alanine resulted in lower levels of l- and d-alanine in the internal pool. With each of these carbon sources, the level of alanine racemase was markedly elevated when compared to glucose-grown cells; thus, with single carbon sources, the concentration of alanine in the pool is inversely related to the specific activity of alanine racemase. These observations support derepression as a possible mechanism that gives rise to higher levels of alanine racemase. Since multiple forms of the alanine racemase were not detected in extracts from E. coli W grown on various carbon sources, it would appear that this type of heterogeneity is not a consideration in interpreting the above results.

Alanine↗
PubMed1972

Mechanism of D-cycloserine action: alanine racemase from Escherichia coli W.

The antibiotic d-cycloserine is an effective inhibitor of alanine racemase. The lack of inhibition by l-cycloserine of alanine racemase from Staphylococcus aureus led Roze and Strominger to formulate the cycloserine hypothesis. This hypothesis states that d-cycloserine has the conformation required of the substrates on the enzyme surface and that l-cycloserine cannot have this conformation. Alanine racemase from Escherichia coli W has been examined to establish whether these observations are a general feature of all alanine racemases. The enzyme (molecular weight = 95,000) has Michaelis-Menten constants of 4.6 x 10(-4)m and 9.7 x 10(-4)m for d- and l-alanine, respectively. The ratio of V(max) in the d- to l-direction is 2.3. The equilibrium constant calculated from the Haldane relationship is 1.11 +/- 0.15. Both d- and l-cycloserine are competitive inhibitors with constants (K(i)) of 6.5 x 10(-4)m and 2.1 x 10(-3)m, respectively. The ratio of K(m)d-alanine to K(i)d-cycloserine is 0.71, and the ratio of K(m)l-alanine to K(i)l-cycloserine is 0.46. Since l-cycloserine is an effective inhibitor, it is concluded that the cycloserine hypothesis does not apply to the enzyme from E. coli W.

Alanine↗
PubMed1971

Mechanism of D-cycloserine action: transport mutants for D-alanine, D-cycloserine, and glycine.

The accumulation of d-alanine and the accumulation of glycine in Escherichia coli are related and appear to be separate from the transport of l-alanine. The analysis of four d-cycloserine-resistant mutants provides additional support for this conclusion. The first-step mutant from E. coli K-12 that is resistant to d-cycloserine was characterized by the loss of the high-affinity line segment of the d-alanine-glycine transport system in the Lineweaver-Burk plot. This mutation, which is linked to the met(1) locus, also resulted in the loss of the ability to transport d-cycloserine. The second-step mutation that is located 0.5 min from the first-step mutation resulted in the loss of the low-affinity line segment for the d-alanine-glycine transport system. The transport of l-alanine was decreased only 20 to 30% in each of these mutants. A multistep mutant from E. coli W that is 80-fold resistant to d-cycloserine lost >90% of the transport activity for d-alanine and glycine, whereas 75% of the transport activity for l-alanine was retained. E. coli W could utilize either d- or l-alanine as a carbon source, whereas the multistep mutant could only utilize l-alanine. Thus, a functioning transport system for d-alanine and glycine is required for both d-cycloserine action and growth on d-alanine.

Alanine↗
PubMed1970

Mechanism of D-cycloserine action: transport systems for D-alanine, D-cycloserine, L-alanine, and glycine.

The accumulation of d-alanine, l-alanine, glycine, and d-cycloserine in Escherichia coli was found to be mediated by at least two transport systems. The systems for d-alanine and glycine are related, and are separate from that involved in the accumulation of l-alanine. d-Cycloserine appears to be primarily transported by the d-alanine-glycine system. The accumulation of d-alanine, glycine, and d-cycloserine was characterized by two line segments in the Lineweaver-Burk analysis, whereas the accumulation of l-alanine was characterized by a single line segment. d-Cycloserine was an effective inhibitor of glycine and d-alanine accumulation, and l-cycloserine was an effective inhibitor of l-alanine transport. The systems were further differentiated by effects of azide, enhancement under various growth conditions, and additional inhibitor studies. Since the primary access of d-cycloserine in E. coli is via the d-alanine-glycine system, glycine might be expected to be a better antagonist of d-cycloserine inhibition than l-alanine. Glycine and d-alanine at 10(-5)m antagonized the effect of d-cycloserine in E. coli, whereas this concentration of l-alanine had no effect.

Alanine↗