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F C Neuhaus

Publications and source records attributed to F C Neuhaus.

At least 37 records · Page 2Linked to original sources
PubMed1985

Biosynthesis of D-alanyl-lipoteichoic acid by Lactobacillus casei: interchain transacylation of D-alanyl ester residues.

Lipoteichoic acid (LTA) from Lactobacillus casei contains poly(glycerophosphate) substituted with D-alanyl ester residues. The distribution of these residues in the in vitro-synthesized polymer is uniform. Esterification of LTA with D-alanine may occur in one of two modes: (i) addition at random or (ii) addition at a defined locus in the poly(glycerophosphate) chain followed by redistribution of the ester residues. A time-dependent transacylation of these residues from D-[14C]alanyl-lipophilic LTA to hydrophilic acceptor was observed. The hydrophilic acceptor was characterized as D-alanyl-hydrophilic LTA. This transacylation requires neither ATP nor the D-alanine incorporation system, i.e., the D-alanine activating enzyme and D-alanine:membrane acceptor ligase. No evidence for an enzyme-catalyzed transacylation reaction was observed. We propose that this process of transacylation may be responsible for the redistribution of D-alanyl residues after esterification to the poly(glycerophosphate). As a result, it is difficult to distinguish between these proposed modes of addition.

Acylation↗
PubMed1984

Biosynthesis of peptidoglycan in Gaffkya homari: processing of nascent glycan by reactivated membranes.

Membranes from Gaffkya homari reactivated by freezing and thawing were used to study the processing events involved in the assembly of both sodium dodecyl sulfate (SDS)-insoluble peptidoglycan (PG) and SDS-soluble PG. The ability to reactivate membranes for the synthesis of these polymers provided an opportunity to monitor those events that are not influenced by wall-linked PG. In G. homari, processing for the formation of cross-links requires the selective actions of DD-carboxypeptidase, LD-carboxypeptidase, and NE-(DAla)-Lys transpeptidase. Time courses of cross-link formation, as measured by the amounts of amidated bisdisaccharide peptide dimer and nonamidated bisdisaccharide peptide dimer, showed a lack of correlation with those for the synthesis of SDS-insoluble PG. SDS-soluble PG, which is significantly cross-linked when synthesized in the absence of penicillin G, was a precursor of the SDS-insoluble PG. In the presence of penicillin G, un-cross-linked SDS-soluble PG was synthesized. This PG was also utilized and processed for the synthesis of cross-linked SDS-insoluble PG after removal of the beta-lactam. This protocol provided a method for separating stages in the synthesis and elongation of PG from those involved in processing. Cross-linkage in the various PG fractions ranged from 0 to 19% in SDS-soluble PG and from 2 to 24% in SDS-insoluble PG. Thus, the results indicated that there is no direct correlation between SDS insolubility and the degree of cross-linkage. Instead, they suggested that additional features may contribute to the insolubility of PG in SDS.

Acetylglucosamine↗
PubMed1983

Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation.

Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1-phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA.

Adenosine Triphosphate↗
PubMed1982

Biosynthesis of peptidoglycan in Gaffkya homari: reactivation of membranes by freeze-thawing in the presence and absence of walls.

The reactivation of membranes from Gaffkya homari for the synthesis of sodium dodecyl sulfate-insoluble peptidoglycan (SDS-insoluble PG) was achieved by successive cycles of freeze-thawing (- 196 versus 25 degrees C). The presence of G. homari walls during this process affected the synthesis of both SDS-soluble (nascent) and SDS-insoluble PG. At two cycles the synthesis of SDS-soluble PG decreased by 70%, whereas that of SDS-insoluble PG increased sevenfold when compared with membranes reactivated in the absence of walls but assayed in the presence of walls. Moreover, at six cycles the lag time for the synthesis of SDS-insoluble PG decreased from 15 min to 5 to 7 min. Walls from G. homari could not be replaced with walls from Bacillus megaterium or cellulose. In addition to these effects, the presence of walls from G. homari or B. megaterium or of cellulose during the incubation of membranes freeze-thawed in the absence of walls increased twofold the amount of SDS-insoluble PG. Reactivated membranes showed greater sensitivities to penicillin (an inhibitor of dd-carboxypeptidase) and d-methionine (an inhibitor of ld-carboxypeptidase) than did isolated membrane-walls. The percentage of cross-linking of the SDS-insoluble PG synthesized by the reactivated system was 34%, a value similar to that observed for the polymer synthesized by isolated membrane-walls. Freeze-thawing membranes and walls together gave a complex with a density different from that of either membranes or walls. Thus, the assembly system for the synthesis and processing of PG was reconstituted in a complex of membranes and walls prepared from the isolated components. Whether this complex has the exact interrelationship between membrane and wall found in the organism has not been established.

Cell Fractionation↗
PubMed1981

Biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei: D-alanyl-lipophilic compounds as intermediates.

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) from Lactobacillus casei contains a poly(glycerol phosphate) moiety that is selectively acylated with D-alanine ester residues. To characterize further the mechanism of D-alanine substitution, intermediates were sought that participate in the assembly of this LTA. From the incorporation system utilizing either toluene-treated cells or a combination of membrane fragments and supernatant fraction, a series of membrane-associated D-[14C]alanyl-lipophilic compounds was found. The assay of these compounds depended on their extractability into monophasic chloroform-methanol-water (0.8:3.2:1.0, vol/vol/vol) and subsequent partitioning into chloroform. Four lines of evidence suggested that the D-alanyl-lipophilic compounds are intermediates in the synthesis of D-alanyl-LTA. First, partial degradation of the poly(glycerol phosphate) moiety of D-alanyl-LTA by phosphodiesterase II/phosphatase from Aspergillus niger generated a series of D-alanyl-lipophilic compounds similar to those extracted from the toluene-treated cells during the incorporation of D-alanine. Second, enzymatic degradation of the D-alanyl-lipophilic compounds by the above procedure gave D-alanyl-glycerol, the same degradation product obtained from D-alanyl-LTA. Third, the incorporation of D-alanine into these compounds required the same components as the incorporation of D-alanine into membrane-associated D-alanyl-LTA. Fourth, the phosphate-induced loss of D-[14C]alanine-labeled lipophilic compounds could be correlated with the stimulation of phosphatidylglycerol synthesis in the presence of excess phosphate. We interpreted these experiments to indicate that the D-alanyl-lipophilic compounds are D-alanyl-LTA with short polymer chains and are most likely intermediates in the assembly of the completed polymer, D-alanyl-LTA.

Alanine↗
PubMed1980

Initial membrane reaction in peptidoglycan synthesis: perturbation of lipid-phospho-N-acetylmuramyl-pentapeptide translocase interactions by n-butanol.

Phospho-N-acetylmuramyl-pentapeptide translocase, the initial membrane enzyme in the biosynthesis of peptidoglycan, requires a lipid microenvironment for function. n-Butanol was reversibly intercalated into membranes to perturb the hydrophobic interactions in this microenvironment in order to define further the role of lipid. In the concentration range for maximal stimulation of enzymic activity (0.12-0.18 M), n-butanol causes a 40% decrease in the fluorescence emission of the dansylated product, undecaprenyl diphosphate-(N epsilon-dansyl)pentapeptide. Since no change in emission maximum occurs below 22 degrees C in the presence of 0.12 M n-butanol, it is concluded that intercalation of this alkanol causes an increase in fluidity. Above 22 degrees C this concentration of n-butanol causes both a decrease in the fluorescence emission and a red shift in the emission maximum. It is concluded that a polarity change as well as fluidity change occurs above 22 degrees C. n-Butanol also causes a significant change in the phase transition experienced by the dansylated lipid product. Thus, it is possible with n-alkanols, e.g. n-butanol, to perturb lipid-translocase interactions resulting in an increase in fluidity in the microenvironment of the enzyme. This change in fluidity correlates with a stimulation of enzymic activity.

1-Butanol↗
PubMed1980

Membrane-wall interrelationship in Gaffkya homari: sulfhydryl sensitivity and heat lability of nascent peptidoglycan incorporation into walls.

Membrane-walls from Gaffkya homari require a specific interrelationship between membrane and wall that functions in the incorporation of nascent peptidoglycan into the preexisting peptidoglycan of the wall. Two different methods were used to inhibit selectively this incorporation process: (i) sensitivity to sulfhydryl reagents and (ii) heat inactivation. Of the sulfhydryl reagents tested, 2.2 mM iodoacetamide inhibited the synthesis of wall peptidoglycan 50%, whereas greater than 100 mM was required to inhibit the synthesis of sodium dodecyl sulfate (SDS)-soluble peptidoglycan. Heat treatment at 37 degrees C (t 1/2 = 5.7 min) inhibited wall peptidoglycan synthesis without affecting SDS-soluble peptidoglycan synthesis. Inhibition of LD-carboxypeptidase by iodoacetamide and heat gave 50% inhibition and t 1/2 values similar to those observed for the incorporation process. Thus, it is suggested that the LD-carboxypeptidase may be one of the enzymes responsible for the sulfhydryl sensitivity and heat lability and that this enzyme may play a role in the relationship between membrane and wall in G. homari.

Carboxypeptidases↗
PubMed1980

Biosynthesis of D-alanyl-lipoteichoic acid: characterization of ester-linked D-alanine in the in vitro-synthesized product.

d-Alanyl-lipoteichoic acid (d-alanyl-LTA) contains d-alanine ester residues which control the ability of this polyer to chelate Mg(2+). In Lactobacillus casei a two-step in vitro reaction sequence catalyzed by the d-alanine-activating enzyme and d-alanine:membrane acceptor ligase incorporates d-alanine into membrane acceptor. In this paper we provide additional evidence that the in vitro system catalyzes the covalent incorporation of d-[(14)C]alanine into membrane acceptor which is the poly([(3)H]glycerol phosphate) moiety of d-alanyl-LTA. This conclusion was supported by the observation that the d-[(14)C]alanine and [(3)H]glycerol labels of the partially purified product were co-precipitated by antiserum containing globulins specific for poly(glycerol phosphate). The isolation of d-[(14)C]alanyl-[(3)H]glycerol from d-[(14)C]alanine.[(3)H]glycerol-labeled d-alanyl-LTA synthesized in the in vitro system indicated that the d-alanine was linked to the poly(glycerol phosphate) chain of the LTA. A comparison of the reactivities of the d-alanine residues of d-alanyl-glycerol and d-alanyl-LTA supported the conclusion that the incorporated residue of d-alanine was attached by an ester linkage. Thus, the data indicated that the in vitro system catalyzes the incorporation of d-alanine covalently linked by ester linkages to the glycerol moieties of the poly(glycerol phosphate) chains of d-alanyl-LTA. New procedures are presented for the partial purification of d-alanyl-LTA with a high yield of ester-linked d-alanine and for the sequential degradation of the poly(glycerol phosphate) moiety substituted with d-alanine of d-alanyl-LTA with phosphodiesterase II/phosphatase from Aspergillus niger.

Alanine↗
PubMed1979

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramyl-pentapeptide translocase.

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by phospho-N-acetylmuramyl (MurN Ac)-pentapeptide translocase (UDP-MurNAc-Ala-gamma DGlu-Lys-DAla-DAla undecaprenyl phosphate phospho-MurNAc-pentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [3H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30 degrees C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of N-oxyl-4',4'-dimethyloxazolidine derivatives of 12- and 16-ketostearic acid intercalated into membranes from this organism define the lower (T1 = 16--22 degrees C) and upper (Th = 30 degrees C) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurNAc-penetapeptide translocase has a significant effect on the catalytic activity of this enzyme.

Cell Membrane↗
PubMed1977

Fluorescent substrate for nascent peptidoglycan synthesis. Uridine diphosphate-N-acetylmuramyl-(Nepsilon-5-dimethylaminonaphthalene-1-sulfonyl)pentapeptide.

The synthesis of UDP-MurNAc-Ala-DGlu-Lys[Nepsilon-dimethylaminonaphthalene sulfonyl (Dns)]-DAla-DAla provides a method for the specific introduction of a fluorescent reporter group into the membrane environment of nascent peptidoglycan synthesis. To assess the degree of perturbation of this environment caused by the introduction of the dansyl substituent, this nucleotide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase (UDP-MurNAc-Ala-gammaDGlu-Lys-DAla-DAla:undecaprenyl phosphate phospho-MurNA-C-pentapeptide transferase) and in the membrane-associated synthesis of nascent peptidoglycan. Phospho-MurNAc-pentapeptide translocase in membrane fragments from Staphylococcus aureus Copenhagen catalyzed the transfer of phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla to undecaprenyl phosphate with a Vmax/Km of 3.8 and a Vmax of 1.6 times the values for UDP-MurNAc-pentapeptide. In the exchange reaction catalyzed by the translocase, the Rmax/Km and Rmax for the dansylated substrate were 1.8 and 0.78 times the respective values for the reference nucleotide. The equilibrium constant for the transfer reaction utilizing UDP-MurNAc-(Nepsilon-Dns)pentapeptide was 5.9 +/- 0.13 compared to 1.1 +/- 0.02 for UDP-MurNAc-pentapeptide. With respect to the proposed reaction model (Pless, D. D., and Neuhaus, F. C. (1973) J. Biol. Chem. 248, 1568-1576), the increase in Keq is consistent with a decrease in the affinity of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla for the translocase. The fluorescence emission maximum of the phospho-MurNAc-(Nepsilon-Dns)pentapeptide moiety of UDP-MurNAc-(Nepsilon-Dns)pentapeptide was blue-shifted from 525 to 495 nm upon transfer from UMP to undecaprenyl phosphate with a 6-fold increase in quantum yield. These spectral changes provided a sensitive and continuous assay for the formation of undecaprenyl diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Dans)-DAla-DAla. The nascent peptidoglycan synthesizing system from Gaffkya homari utilized the dansylated nucleotide with a Vmax/Km of 0.05 and a Vmax of 0.10 times the values for UDP-MurNAc-pentapeptide. These results demonstrate that phospho-MurNAc-Ala-DGlu-Lys(Nepsilon-Dns)-DAla-DAla linked to the undecaprenyl phosphate will serve as a precursor for the synthesis of nascent peptidoglycan and that the dansyl moiety will report on the membrane environment it experiences during this synthesis.

Cell Membrane↗
PubMed1977

Biosynthesis of spin-labeled peptidoglycan: spin-spin interactions.

Membrane preparations from Gaffkya homari catalyzed the in vitro biosynthesis of soluble uncross-linked spin-labeled peptidoglycan, a uniformly labeled polynitroxide, from the spin-labeled nucleotide UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-2,2,5,5-tetramethyl-1-pyrrolin-1-oxyl-3-carbonyl)-DAla-DAla (I) and UDP-GlcNAc. Soluble spin-labeled peptidoglycan was separated from membrane fragments and its spin-labeled precursor by centrifugation and gel filtration. The molecular weight distribution of the polymer was examined by agarose gel filtration. Spin-labeled [14C]peptidoglycan was polydisperse with a peak of radioactivity corresponding to a molecular weight of 5.0 X 10(5). The electron spin resonance spectrum of spin-labeled peptidoglycan was extensively broadened by spin-spin exchange interactions. These interactions were modified by changes in temperature, reduction by ascorbate, hydrolysis by lysozyme, and complexation with the antibiotic, vancomycin. Spin-spin exchange was reduced or eliminated in spin-labeled peptidoglycan by the random reduction of free radicals by ascorbate. A rotational correlation time of 0.37 ns was calculated for the probe in partially reduced spin-labeled peptidoglycan. This compares to a correlation time of 0.13 ns for the substrate (I). Raising the temperature increases spin-spin exchange line broadening. No transition points were observed for spin-labeled peptidoglycan as measured by this method. Degradati on of spin-labeled peptidoglycan by lysozyme eliminated the observed spin-spin exchange and yielded products with a mobility similar to I. Complexation of spin-labeled peptidoglycan with vancomycin resulted in both pronounced free-radical immobilization and a decrease in spin-spin exchange. The exchange effects are consistent with distance measurements in molecular models for peptidoglycan.

Binding Sites↗
PubMed1977

Growth inhibition of Escherichia coli W by D-norvalyl-D-alanine: an analogue of D-alanine in position 4 of the peptide subunit of peptidoglycan.

Position 4 analogues of d-alanine in the peptide subunit of uridine 5'-diphosphate-N-acetylmuramyl-Ala(1)-dGlu(2)- Lys(3)-dAla(4)-dAla(5) have a significant inhibitory effect on penicillin-sensitive peptidoglycan synthesis in Gaffkya homari (C. V. Carpenter, S. Goyer, and F. C. Neuhaus, 1976). The specificity profile of this in vitro system has been used as a basis for designing analogues with potential antibacterial activity. To circumvent the specificity determinants exerted by d-alanine:d-alanine ligase (adenosine 5'-diphosphate), attention was directed to dd-dipeptides of the type d-alanyl-analogue-d-alanine as a method for incorporating analogues into position 4 of the peptide subunit in vivo. Of the three dipeptides, dAbu-dAla, dNva-dAla, and dVal-dAla, only dNva-dAla (5 x 10(-4) M) inhibited the growth of Escherichia coli W in the presence of 5 x 10(-6) M d-cycloserine. This concentration of d-cycloserine did not inhibit growth, but it potentiated the bactericidal activity of the dipeptide. The lack of antibacterial activity observed with dAbu-dAla and dVal-dAla was correlated with the poor ability of these dipeptides to be taken up via the dipeptide transport system of this organism. Prevention of lysis induced by dNva-dAla plus d-cycloserine by certain dipeptides and not by others supported this correlation. It is proposed that the d-norvalyl residue of the dipeptide is incorporated in vivo into position 4 of the peptide subunit of peptidoglycan, and that this subunit is not an effective substrate in the reaction(s) catalyzed by the penicillin-susceptible enzyme(s) of cross-linked peptidoglycan synthesis.

Alanine↗
PubMed1976

Steric effects on penicillin-sensitive peptidoglycan synthesis in a membrane-wall system Gaffkya homari.

Residues 4 and 5 of the pentapeptide moiety, R-Ala1-DGlu2-Lys3-DAla4-DAla5, of peptidoglycan play an important role in the donor phase of cross-linked glycan synthesis. To assess the role of these residues in this phase, a series of UDP-MurNAc-peptides were biosynthesized with residues 4 and 5 replaced singly by either D-alpha-amino-n-butyric acid, D-norvaline, or D-valine. The six nucleotides were compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla (reference) in nascent (penicillin-insensitive) peptidoglycan synthesis and in penicillin-sensitive peptidoglycan synthesis. The synthesis of penicillin-sensitive peptidoglycan is catalyzed by membrane-walls isolated from Gaffkya homari and would appear to require the concerted action of transglycosylase and transpeptidase. The membrane-wall system shows a high degree of discrimination for the steric substituents, -CH3 and -CH2CH3, in residue 4. For example, for UDP-MurNAc-Ala-DGly-Lys-DAbu-DAla and -Ala-DGlu-Lys-DAla-DAbu, Vmax/km is 0.19 and 0.95 and Vmax is 0.03 and 0.52, respectively, of the value for the reference nucleotide. In contrast, for the synthesis of nascent peptidoglycan with these nucleotides Vmax/Km is 0.75 and 0.80, and Vmax is 0.71 and 1.0, respectively, of the value for the reference nucleotide. This trend was also illustrated with the other nucleotides in the time course experiments. These results indicate that the penicillin-sensitive enzyme(s), presumably the transpeptidase, has a higher degree of specificity in the donor phase for D-alanine in residue 4 than for D-alanine in residue 5 in the cross-linking stage of peptidoglycan synthesis.

Acetylglucosamine↗
PubMed1976

Biosynthesis of peptidoglycan in Staphylococcus aureus: incorporation of the Nepsilon-Ala-Lys moiety into the peptide subunit of nascent peptidoglycan.

UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla was isolated from extracts of Staphylococcus aureus Copenhagen. This nucleotide accumulated in media deficient in glycine. To establish its role in peptidoglycan biosynthesis, the nucleotide-hexapeptide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase and in the membrane-catalyzed nascent peptidoglycan-synthetizing system. In the exchange reaction catalyzed by the translocase, the Rmax and Rmax/Km are 1.79 muM/min and 4.47 X 10(-2)/min, respectively, for UDP-MurNAc-pentapeptide and 1.81 muM/min and 4.46 X 10(-2)/min, respectively, for UDP-Mur-NAc-hexapeptide. In the synthesis of nascent peptidoglycan, the Vmax is 1.8 muM/min X 10(-2) for both the nucleotide-hexapeptide and -pentapeptide. The Vmax/Km is 5.6 X 10(-4) and 4.3 X 10(-4)/min for the nucleotide-pentapeptide and -hexapeptide, respectively. Schleifer, Hammes, and Kandler (Adv. Microb. Physiol. in press) observed that growth of S. aureus Copenhagen on a glycine-poor medium results in a peptidoglycan structure in which 20% of the lysine residues are substituted at the epsilon-amino group by L-alanine residues that do not participate in interpeptide bridge information. The in vitro studies demonstrate that UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla is a possible precursor of the Nepsilon-Ala-Lys moiety.

Acetylglucosamine↗
PubMed1975

Initial membrane reaction in the biosynthesis of peptidoglycan. Spin-labeled intermediates as receptors for vancomycin and ristocetin.

Phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla:undecaprenyl phosphate, phospho-MurNAc-pentapeptide transferase) catalyzes the initial membrane reaction in the biosynthesis of peptidoglycan. The spin-labeled nucleotide, UDP-MurNAc-Ala-DGlu-Lys (Nepsilon-2,2,5,5-tetramethyl-N-oxyl-pyrroline-3-carbonyl)-DAla-DAla, was used as a substrate by this enzyme for the synthesis of membrane-associated undecaprenyl-diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Tempyo)-DAla-DAla. The spin-labeled substrate and product complex with the antibiotics vancomycin and ristocetin. The association constants for the spin-labeled nucleotide are 6.2 times 10(5) and 6.2 times 10(4) M-1 for vancomycin and ristocetin, respectively. The association constants for the spin-labeled lipid intermediate are 3.0 times 10(4) and 2.1 times 10(4) M-1 for vancomycin and ristocetin, respectively. These results indicate that the acyl-DAla termini of membranes-associated spin-labeled undecaprenyl-diphosphate-MurNAc-pentapeptide are accessible to vancomycin and ristocetin and that the association constants are smaller than those determined for the corresponding antibiotic spin-labeled UDP-MurNAc-pentapeptide complexes.

Binding Sites↗