Eur. J. Biochem. 195, 763-769 (1991) 0 FERS 1991

001429569100092J

Dependence of lysozyme-catalysed solubilization of Proteus mirabilis peptidoglycan on the extent of 0-acetylation Claude DUPONT and Anthony John CLARKE Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Microbiology, University of Guelph, Ontario, Canada

(Received October 2, 1990) - EJB 90 1181

The degree of peptidoglycan 0-acetylation in 14 strains of Proteus mirabilis has been accurately determined by a procedure which employs the quantitation of mild-base-released acetic acid by HPLC, and the estimation of peptidoglycan concentration by cation-exchange amino acid analysis. The /3-~-N,6-O-diacetylmuramyl content of all isolated and purified peptidoglycans was ranged 20- 52.8%, relative to the total muramic acid concentration. Each of the 0-acetylated peptidoglycans was found to be resistant to solubilization by both human and hen egg- white lysozymes and for hen egg-white lysozyme, the extent of this resistance was dependent upon the degree of 0-acetylation. The steady-state parameters, K, and V, for the hen-egg-white-lysozyme-catalysed solubilization of various peptidoglycan preparations were determined at pH 6.61 and 25°C. Values of K , for the different peptidoglycan samples were found to increase with increasing 0-acetylation, whereas with V no such relationship appeared to exist. An increase in the overall change in the standard Gibbs free energy of activation [d(dG*)], a consequence of increasing 0-acetylation, was observed, and is shown to result from the weaker affinity of the enzyme for the modified substrates.

Peptidoglycan is a heteropolymer of distinct composition and structure, associated uniquely with eubacterial cell walls. Proteus mirabilis peptidoglycan is comprised of equimolar amounts of GlcNAc, N-acetylmuramic acid (MurNAc), L- alanine, D-glutamic acid, L-meso-diaminopimelic acid and D- alanine. The cross-linking of neighbouring peptides is direct (chemotype Aly) and involves the meso-diaminopimelic acid and D-alanyl residues. The extent of cross-linking (33%) is typical of most Gram-negative bacteria [l]. Thus, the peptidoglycan of P. mirabilis is characteristic of the Enter- obacteriaceae but for one notable and significant difference, the presence of extensive 0-acetylation [2]. Peptidoglycan 0- acetylation occurs at the 6-hydroxyl group of MurNAc resi- dues producing the corresponding N,O-diacetylmuramyl de- rivatives, and this modification confers resistance to the hy- drolytic action of many muramidases, including hen egg-white lysozyme (HEWL) and human lysozymes [3 - 61.

The phenomenon of peptidoglycan 0-acetylation was first observed independently by two different groups of researchers over 30 years ago [7, 81, and its role in conferring lysozyme resistance was demonstrated soon after [6]. Since then, 0- acetylation of peptidoglycan has been observed in a diverse group of bacteria which includes some important pathogens, both Gram positive (e.g. Staphylococcus aureus [9, 101) and Gram negative (e. g. Neisseria gonorrhoeae [3] and P. mirabilis [2]). A strain-related difference in lysozyme sensitivity with degree of 0-acetylation for gonococcal peptidoglycan has been established [4, 11, 121. Peptidoglycan isolated from 13

Corrapondence to A. J. Clarke, Department of Microbiology,

Abbreviations. HEWL, hen egg-white lysozyme; HML, human

Enzymes. Lysozyme (EC 3.2.1.17); pronase (EC 3.4.24.4).

University of Guelph, Guelph, Ontario, N1G 2W1, Canada

milk lysozyme; MurNAc, N-acetylmuramic acid.

strains of N . gonorrhoeae and characterized by 34- 52% 0- acetylation, was shown to be resistant to hydrolysis by HEWL to the extent of 34-59% [12]. On the other hand, O-acetyl- deficient peptidoglycan, isolated from N . gonorrhoea RD5 was completely solubilized and degraded to low-molecular-mass fragments. In the study, resistance was defined as the percent- age increase in size compared to tetrameric peptidoglycan subunits (GlcNAc-MurNAc-Xaa4).

The biological significance of peptidoglycan 0-acetylation with respect to microorganisms is still unknown, but the per- sistence of high-molecular-mass peptidoglycan fragments circulating in a host organism has been shown to have serious pathobiological and pathophysiological consequences. In man, these include activation of complement, modulation of blastogenesis, pyrogenicity and possibly, arthrogenicity (for a recent review see [13]). Rosenthal and co-workers unequivo- cally demonstrated in rat-model studies that highly 0- acetylated gonococcal peptidoglycan was more effective in inducing an arthritic condition than either the non-0- acetylated material of strain RD5 or chemically de-0- acetylated peptidoglycan [I 11. These investigators thus pro- vided strong evidence suggesting that the persistence of lysozyme-resistant high-molecular-mass fragments, which are a consequence of extensive 0-acetylation, may be important for optimal induction and expression of arthropathic activity.

Whereas lysozyme resistance has been attributed to peptidoglycan 0-acetylation, an explicit correlation between this modification and the kinetics of lysozyme action has yet to be established. In the present study, we report the kinetic parameters for the lysozyme-catalyzed solubilization of a number of peptidoglycans isolated from a variety of P. mirabilis strains, and demonstrate the adverse effect of 0-

764

acetylation on the affinity (K,) of the enzyme for the insoluble substrate.

MATERIALS AND METHODS

Bacteria

Eight strains of P. mirabilis were obtained from the American Type Culture Collection (ATCC 7002,12453,14273, 25933, 29245, 33583, 33659 and 43057). Toronto-General- Hospital isolates (TGH 7341, 9047) and type strains of the Kauffmann [14] scheme, F491 (03) and F16 (05) were kindly provided by Dr. J. L. Penner, Department of Medical Micro- biology, University of Toronto. Two further isolates (strains 19 and GB8) were obtained from Dr. J. Gmeiner, Technische Hochschule Darmstadt, Federal Republic of Germany. Escherichia coli K12 (CSH4) was provided by Dr. J. Wood, Department of Microbiology, University of Guelph. All bac- teria were maintained on nutrient-agar slants at 4°C.

Enzymes and biochemicals

Mutanolysin, human milk lysozyme (HML), HEWL, GlcNAc, MurNAc, muramic acid, chitin and SDS were pur- chased from Sigma Chemical Co., St. Louis, MO, while Boehringer Mannheim Canada, Laval, Que, supplied the pro- nase. All other chemicals were obtained from Fisher Scientific, Toronto, Ontario, and were of reagent grade.

Preparation qf’insoluble peptidoglycan

Cultures (1 1) of the various microorganisms were grown in nutrient broth supplemented with 30 mM glucose at 37°C under forced aeration. After 4.5 h incubation, the cells (A578 0.80 - 0.90) were harvested by centrifugation (Sorvall RC-2B, DuPont Sorvall) at 8000 x g and 4°C for 15 min, washed twice with 10 mM sodium phosphate buffer, pH 6.5, then suspended in 20 ml water (pH 5.5 - 6.0). Insoluble peptidoglycan was extracted from whole cells employing the procedure of Hoyle and Beveridge [I 51. Cell suspensions were added dropwise to an equal volume of 8 % (mass/vol.) SDS in 10 mM sodium phosphate, pH 6.5, at 100°C. After the cells had all been added, the SDS extraction at 100°C was continued for 3.5 h under reflux. After cooling to ambient temperature and stir- ring overnight, the SDS-insoluble material was collected by ultracentrifugation (Beckman L5-50, Beckman Instruments, Canada) at 160000 x g for 60 min at 20°C. The pellet was washed three times by centrifugation with dilute HCI, pH 5.5 and resuspended in 10 ml 10 mM Tris/HCl buffer, pH 6.8. These crude peptidoglycan preparations were treated with 0.2 mg/ml (final concentration) pronase (heated for 2 h at 60 ’C) for 60 min at 60 “C. The peptidoglycan was re-extracted with 4% (final concentration) SDS in 10 mM sodium phos- phate buffer, pH 6.5, at 100°C for 45 min. The insoluble ma- terial was collected and washed by centrifugation three times as described above, and finally lyophilized. Prior to use, the isolated and purified peptidoglycan was suspended in 0.1 YO (mass/vol.) sodium azide at a final concentration of 3 mg/ml and stored at 4°C. In a separate experiment to assess the stability of U-acetylated peptidoglycan during the isolation procedure, insoluble peptidoglycan was extracted as above with 4% SDS in both water and 5 m M sodium phosphate buffer ranging in pH 4.5-8.5.

Release and quantitation of 0-acetate

Aliquots (200 pl) of peptidoglycan suspensions or model compounds (chitin, GlcNAc, MurNAc) in 0.1 YO sodium azide were incubated overnight at ambient temperature with an equal volume of either 40 mM NaOH or 50 mM sodium phos- phate buffer, pH 6.5. The peptidoglycan was collected by centrifugation at 160000 x g for 10 min at 4°C using a Beckman Airfuge, and the pellet was washed once by centrifugation with 200 p1 0.1 O/O sodium azide. The super- natants were pooled and filtered through a Millipore HA 0.45 pm membrane (Millipore Ltd, Mississauga, Ontario). Detection and quantitation of released acetate was ac- complished by HPLC employing an HPX-87H organic-acid column (Bio-Rad Laboratories Inc., Mississauga, Ontario). Samples of the pooled and filtered supernatants (250 pl) were injected onto the column at 45’C with 10 mM H2S04 serving as eluent at a flow rate of 0.50 ml/min. Detection of acetate was by monitoring the absorbance of the column effluent at 210 nm.

Enzyme kinetics

The turbidometric assay of Hash [16] was employed to assess the susceptibility of the peptidoglycan preparations to various muramidases and determine the kinetic parameters. Briefly, appropriately diluted and evenly suspended (sonicat- ed) aliquots (200 pl) of insoluble peptidoglycan were added to 550 p1 of 100 mM phosphate buffer, pH 6.61, in a 1-cm path length glass cuvette, to give the desired concentration of sub- strate (final ionic strength of 130 mM). Hydrolytic reactions were initiated by adding 50 p1 enzyme solutions (250-2500 units/ml), and the decrease in turbidity at 600 nm was monitored with time. The initial velocity (v) was obtained from linear portions of the graphs during the early stages of depolymerization (within the first three minutes) and values for K, and V were obtained from computerized least-squared fits of the Eadie-Hofstee equation to the data.

Aizalyt ical methods

Peptidoglycan concentrations were determined by amino acid analysis using a Beckman System Gold amino acid analyzer with post-column ninhydrin detection (Beckman In- struments Ltd, Toronto, Ontario). Samples of peptidoglycan (150 pg) were hydrolysed in vucuo with 4 M HC1 at 110°C for 18 h. Acetate quantitation by HPLC was performed on a Beckman system comprised of two model l l0B pumps, a model 167 dual-channel rapid-scanning ultraviolet/visible de- tector, a model 406 analog interface, an IBM-XT computer controller with Beckman Gold chromatography software and a Bio-Rad HPLC column heater. Turbidometric measure- ments were made using either a Beckman model DU8 or a Varian Model 2290 recording ultraviolet/visible spectro- photometer.

RESULTS

Extent of peptidoglycan 0-acetylation

The HPLC chromatograms of released acetate indicated that mild base treatment with 20 mM NaOH removes all 0- linked acetate from isolated and purified peptidoglycan, while leaving N-acetyl moieties intact. No released acetate was de- tected from samples of similarly treated non-U-acetylated E.

765

- 1 0 0 -

v,

a 8 0 : 60 c3

Table 1. Effect of pH on the extent of 0-acetylation recovery during pep t idog ly can isolation Isolation buffer, 4% SDS in 5 mM sodium phosphate. The percentage 0-acetylation was calculated assuming a molecular mass of 942 Da for one peptidoglycan subunit of GlcNAc-MurNAc-Xaa4. Results expressed mean f SD, n = 3

I. I Isolation Base-released acetic acid 0-Acetylation buffer pH

4.3 5.0 5.6 6.2 6.8 7.4 8.9 8.0"

nmoles . mg peptidoglycan- ' 463 452 41 8 515 41 5 43.5 25.2 29.8

% recovery

87.6 (4.77) 85.4 (2.50) 79.1 (6.32) 97.6 (4.81) 78.4 (5.27)

8.24 (1.53) 4.77 (1.69) 5.66 (2.70)

a HzO.

60

c 0 .- 4

- CI 40 x Q, 0 Q

0

x

4

' 20

0

- P. mirabilis Strain Fig. 1. Degree ofpeptidoglycan 0-acetylation in strains of P. mirabilis expressed as a percentage of the amount of acetic acid released by mild base hydrolysis and detected by HPLC analysis, relative to the concentration of N-acetylmuramic acid determined by amino acid analysis. Standard deviations were no higher than 2% for each deter- mination (n = 3)

coli peptidoglycan, or from any of the model compounds, chitin, GlcNAc or MurNAc. Moreover, no other peptidoglycan degradation components, including muramic acid and its derivatives [17], were found to elute with acetate from the HPLC column under the conditions employed. The sensitivity of detection using this rapid HPLC method was greater than 5 nmoles acetate. Accurate estimations of peptidoglycan 0-acetylation, however, required the mainten- ance of acidic conditions during the SDS-extraction procedure to prevent premature base hydrolysis (Table 1). Moreover, the results of the pH-dependence study indicate that the common practise of employing unbuffered SDS-extraction protocols is not adequate for the isolation of intact 0-acetylated peptidoglycan. This phenomenon was exploited for the iso- lation of partially de-0-acetylated peptidoglycan (see below).

7 I

B

I 0.0 5.0 10.0 15.0

Time (min) Fig. 2. Muramidase solubilization of peptidoglycan (PG) isolated and purified from ( A ) P. mirabilis 19 (closed symbols) and E. coli K12 (CSH4) (open symbols) and ( B ) P. mirabilis ATCC 29245. Peptidoglycan (0.75 mg/ml) in 65 mM sodium phosphate buffer, pH 6.61, was incubated at 25°C with (0, 0 ) HEWL (2.5 U); ( A , A) HML (2.5 U); ( W ) mutanolysin (250 U). The decrease in A600, monitored with time, is expressed as a percentage of an appropriate control which lacked muramidase addition

The extent of peptidoglycan 0-acetylation, determined by the HPLC assay for the various bacterial strains, is illustrated in Fig. 1. All of the 14 strains of P. mirabilis tested had 0- acetylated peptidoglycan levels greater than 20% (molar ratio). Strain 19, provided by J. Gmeiner, possessed the hghest level of 0-acetylation (52.8%). This percentage is lower than that reported by Gmeiner (60.6% [18]) and probably reflects differences in the analytical techniques used. Hitherto, autoradiography of either paper or thin-layer chromatograms was used to estimate the percentage of 0-acetylation of peptidoglycans following their digestion with Chuluropsis N,O-diacetylmuramidase.

Susceptibility of 0-acetylpeptidoglycan to digestion by muramidase

SDS-insoluble peptidoglycan from both P. mirabilis 19 (52.8% 0-acetylated) and E. coli K12 (CSH4) was treated at pH 6.61 with three different muramidases, HEWL (2500 units), HML (2500 units) and mutanolysin (250 units). The degree of solubilization was monitored turbidometrically with time, and representative time-course curves are presented in Fig. 2A. All three enzymes completely solubilized E. coli peptidoglycan within 3 min of enzyme introduction, whereas only mutanolysin, a B-~-N,6-O-diacetylmuramidase [19], was effective in solubilizing the P. mirabilis 19 peptidoglycan prep- aration. After 15 rnin incubation, HEWL and HML only solubilized P. mirabilis 19 peptidoglycan 19% and 22%, re- spectively. Upon prolonged incubation (24 h), HEWL con- tinued to completely solubilize P. mirabilis peptidoglycan, albeit at a very slow rate, while HML was inactive. Analogous results were obtained (Fig. 2B) with the insoluble

766

1

r 0

, . r

HML i

Time (min) Fig. 3. Dependence of rute of nzurumi~use-cutulysed solubilization of peptidoglycan ( P G ) on extent of 0-acetylution. Native peptidoglycan (0.75 mgiml), isolatcd from P. mirabilis, ( 0 ) strain 19 (52.8% 0- acetylated), ( W ) strain ATCC 29245 (31.6% 0-acetylated), (+) strain ATCC 43057 (27.494 0-acctylated), and chemically de-0-acetylated P. nzirahilis peptidoglycan (0.75 mg/ml) with, (0) 13.6%; (3) 8.54%; (0) non-0-acetylated peptidoglycan was treated with either HEWL or HML at pH 6.61 and 25 C. The decrease in A600 was monitored with time and expressed as a percentage of the control peptidoglycan solutions

peptidoglycan isolated from P. mirabilis ATCC 29245 (31.6% 0-acetylated). However, the rates of HEWL-catalysed hy- drolysis of the two peptidoglycan preparations, which are 0- acetylated to different extents, appear to be different under similar conditions. This was not apparent in the case of HML as both samples of peptidoglycan remained similarly resistant to the hydrolytic action of the enzyme.

The apparent dependence of HEWL activity on the extent of peptidoglycan 0-acetylation was further investigated by isolating peptidoglycan from P. mirahilis strains differing in their 0-acetyl content. In addition, samples of partially and fully de-0-acetylated P. mirabilis peptidoglycan were chemi- cally prepared by either mild base hydrolysis for a short period of time at 4 C or peptidoglycan isolation employing unbuffered 4%) SDS. These different peptidoglycan prep- arations were incubated with both HML and HEWL as de- scribed above, and typical progress curves are presented in Fig. 3. It is clear that with HEWL, decreasing the extent of 0-acetylation results in an increase in the rate of solubil- ization. In contrast, none of the naturally occurring peptidoglycans isolated from the various P . mirabilis strains were more susceptible to HML. This enzyme was able to catalyze partial solubilization (60 - 70%) of peptidoglycan only after limited chemical de-0-acetylation of the substrate. Complete HML-catalysed solubilization of peptidoglycan re- quired the removal of entire 0-acetyl moieties.

Dependence OJ'HE WL kinetic. parameters on 0-acetylation

Peptidoglycan was isolated and purified from selected P. mirabilis strains which represented a range of 0-acetyl con-

tent, and together with chemically de-0-acetylated material was used as substrate for the determination of the respective kinetic constants, K, and V of HEWL. v values for the HEWL-catalysed solubilization of five concentrations (0.125 - 1.5 mg/ml) of the various peptidoglycan samples were determined (in triplicate) at pH 6.61 (ionic strength of 130 mM) and used to calculate the steady-state parameters. Under these conditions, substrate inhibition of HEWL by insoluble peptidoglycan is minimized [20]. The data, presented in Table 2, clearly reflect a dependence of the pseudo-second- order rate constant (V/Km) of HEWL on the degree of peptidoglycan 0-acetylation. This is illustrated in Fig. 4, where the enzymatic efficiency increases to a maximum with complete de-0-acetylation of peptidoglycan. Interestingly, a linear relationship appears to exist between log ( V/K,) and the degree of 0-acetylation (Fig. 4, inset) suggesting that the dependence of catalytic efficiency on 0-acetylation obeys the Taft equation [21]

log k = @*o* + hE,. (1) where Q*, o*, 6 and E, are the polar reaction, polar substituent, steric reaction and steric substituent constants, respectively. Plotting values for K, and V against the percentage O-acety- lation separately (Fig. 5 ) reveals that K, is adversely affected by the modification, whereas no such trend is apparent for V.

An increase in the overall standard Gibbs free energy of activation [d(dG*)] was also observed with increasing 0- acetylation of peptidoglycan (Table 2). These values are re- adily calculated from the relative values of V/Km for the peptidoglycan in question, and for the de-acetylated substrate, using the expression below :

where ( V/K,,Jl and ( V/Km), are the kinetic constants for the 0-acetylated and de-0-acetylated substrates, respectively.

DISCUSSION

We have examined purified samples of peptidoglycan from a range of P. mirabilis strains for the degree of 0-acetylation, and have attempted to correlate these findings to the extent of HEWL resistance. Accurate quantitation of peptidoglycan 0-acetylation was facilitated by employing HPLC procedures for both acetate and amino-sugar analysis. All of the strains examined possessed peptidoglycan which was both 0- acetylated and HEWL resistant. The degree of 0-acetylation ranged between 20% and 52.8%, which was similar to that observed in other bacteria.

Approximately 50% of the muramyl residues of P. vulgaris PI8 peptidoglycan have been shown to be N,O-diacetylated [22]. With N. gonorrhoea, Rosenthal and co-workers [I31 de- tected 34- 52% 0-acetylation of peptidoglycan with a pre- ponderance of strains possessing above 40% 0-acetylation. The muramyl residues of S. aureus peptidoglycan are about 60% 0-acetylated [23], but this value appears to increase the approximately 82% when cells are cultured in the presence of the bacteriostatic drug, chloramphenicol[24]. Indeed, being a maturation process, the extent of 0-acetylation is observed to increase during the stationary phase [24].

Working with a HEWL-resistant mutant of Micrococcus lysodeikticus, Brumfitt and co-workers [6, 81 were the first to suggest that 0-acetylation of peptidoglycan confers resistance to the hydrolytic action of muramidases. However, discrep-

767

Table 2. Dependence ofthe kinetic parameters of HE WL-catalysed hydrolysis of isolated P. mirabilispeptidoglycan on the degree of 0-acetylation 0-Acetylation is expressed as a percentage of the acetate released upon mild base hydrolysis compared to that released after total hydrolysis of muramic acid. Corrected HEWL kinetic parameters were calculated after adjusting the substrate concentrations to reflect the putative blockage of the binding sites on N,O-diacetylmuramyl residues. K, is expressed as the peptidoglycan monomers (GlcNAc-MurNAc-Xaa,; molecular mass 942 Da). P. mirabilis strains 19 (43.6 and 8.54% 0-acetylation), ATCC 7002 and F491 were partially de-0-acetylated by mild base hydrolysis. 'De-0-acetyl' includes the completely de-0-acetylated pooled ATCC 7002, ATCC 29245, ATCC 33659, ATCC 43057 and GB8

P. mirubilis Peptidoglycan HEWL kinetic parameters HEWL kinetic parameters strain 0-acetylation

K m V VIK, A ( A G * ) K, VIKm

% mM 1 0 3 . A600 103 . kJ . mol-' mM 103 . A600 . min-' . mM-l . mM-l

19 52.8 1.22 2.26 1.85 12.0 0.550 4.11 19 43.6 0.780 1.69 2.26 11.6 0.440 3.84 ATCC 7002 43.6 0.895 4.39 5.20 9.45 0.505 8.69 ATCC 7002 36.3 0.580 19.4 33.4 4.89 0.370 52.5 ATCC 33583 31.9 0.385 11.7 30.4 5.10 0.262 44.7 ATCC 29245 31.6 0.365 6.25 17.1 6.52 0.250 25.1 ATCC 33659 24.7 0.300 7.86 26.2 5.48 0.226 34.8 ATCC 12453 24.1 0.186 18.8 101 2.15 0.141 133 F491 15.4 0.129 29.8 231 2.15 0.109 274 19 8.54 0.105 11.5 109 0.1 1 0.0964 119 De-0-acetyl 0.001 0.0563 13.6 242 1.96

250.0 ' 3.0 1-1

200.0

150.0

\ 100.0

E 50.0

0.0

E Y

x U

>

5 2 . ~ ~ ~ : 1.0

1

0.0 0 20 40 60

% 0-Acetylation .

0 20 40 60 % 0-Acetylat ion

Fig. 4. Dependence of efficiency ofthe HEWL-catalysed soluhilization of P. mirabilis peptidoglycan on the degree of 0-acetylation. Apparent second-order rate constants were determined using the steady-state parameter (Table 2) obtained from Eadie-Hofstee plots of the initial velocity of solubilization versus the peptidoglycan concentration. v,,,/ K, expressed as . min . mM. Inset. Semi-log plot of the apparent second-order rate constants as a function of degree of peptidoglycan 0-acetylation. Closed symbols, native peptidoglycan; open symbols, chemically de-0-acetylated peptidoglycan preparations

ancies later appeared in the literature concerning this inhi- bition. For example, prior base hydrolysis of 0-acetylated peptidoglycans isolated from P. vulgaris, N . perfava and Pseudomonas alcaligenes appeared to have little effect on lysozyme sensitivity [25]. It is quite possible that the apparent lack of increased HEWL activity following mild base hydroly- sis was related to the method of peptidoglycan purification because residual fragments of lipoproteins may have remained linked to the polymers, thereby providing further muramidase resistance. With respect to our observations concerning purified human lysozyme, resistance is absolute since after prolonged incubation various 0-acetylated peptidoglycan

preparations remained virtually insoluble as detected by tur- bidometry. This is analogous to the situation observed with purified human leukemic polymorphonuclear lysozyme acting on 0-acetylated gonococcal peptidoglycan [5].

The kinetic data obtained with HEWL indicate that the peptidoglycans from different strains of P. mirabilis can differ markedly in their susceptibility to solubilization, and that the susceptibility correlates with the degree of 0-acetylation. Similar results have been obtained with gonococcal peptidoglycan [4, 121. Although steady-state parameters were not measured, the previous studies examined a broad range of N . gonorrhoea strains and related HEWL resistance to 0- acetylation, where resistance was expressed as the percentage of the peptidoglycan which was larger in size than disacchar- ide-peptide tetramers. Hence, it may be misleading to draw any close parallels between the current and previous data since different events were monitored, that is, solubilization versus resistance.

Extending the results of the present kinetic study, it is evident that A(AG*) of the HEWL-catalysed hydrolysis of peptidoglycan is dependent on the degree of 0-acetylation. We are presently not in a position to provide a complete explanation of the mechanism for this phenomenon, but infor- mation from the comprehensive literature concerning the structure and functional relationship of HEWL does provide some insight. The decreasing rates of peptidoglycan hydrolysis observed with increasing 0-acetylation could arise from het- erogeneity in the local distribution of the acetyl moieties, with HEWL binding and hydrolysis only occurring at non-0- acetylated positions. In this scheme, fewer potential sites for hydrolysis would be available to the enzyme on highly 0- acetylated peptidoglycan, resulting in a lower effective concen- tration of substrate. Alternatively, HEWL binding may occur at any site along the glycan backbone, but with weaker affin- ities at sites of 0-acetylation. Analysis of the X-ray crystal- lographic structures of HEWL-inhibitor complexes [26] indi- cate that contacts are made between the enzyme and C6 hy- droxyl groups of muramyl residues. Thus, it is plausible that acetyl modification at these positions either precludes or con- strains HEWL binding. This is further supported by kinetic

768

30.0

20.0

X U E 10.0

>

0.0 1.6

- 1.2 2

0.8

Y 0.4

0.0

E

€

B

0.0 0 5 J I

m-l.o 7lP"$ 9 - -1.5- 0 20 40 60

0 20 40 60 % 0-Acety la t ion

Fig. 5 . Dependence of' i A J V and ( B ) K, on the degree of 0-acetylation for the H E WL-cataljsed solubilization of P. mirabilis peptidoglycan. Inset. Semi-log plot of K,,, versus the percentage of U-acetylation, indicating the relationship between HEWL-substrate binding affinity and degree of substrate modification. Closed symbols, native peptidoglycan isolated from different P. mirabilis strains; open sym- bols, chemically de-0-acetylated peptidoglycan preparations. V,,, values expressed as lo3 . A600 . min-'

studies on the rates of solubilization for the various peptidoglycan derivatives. While caution should be exercised when interpreting the steady-state parameters determined from the solubilization of an insoluble substrate, an analysis of the relationship between the rate of solubilization and ex- tent of 0-acetylation indicates that it is dependent upon K, (Fig. 5) . An increase in K,,, with increasing 0-acetylation would be expected to arise if the number of available binding sites on the insoluble substrate were decreased by modifi- cation. However, correcting for the putative decrease in effec- tive substrate concentration as a result of muramyl O-acety- lation suggests that the role of 0-acetylation in HEWL inhi- bition is more complex than simply denying enzyme accessi- bility. By decreasing each substrate concentration by a factor determined by the extents of 0-acetylation, and re-calculating the steady state parameters (Table 2), we find that pseudo- second-order rate constants are still dependent upon the modi- fication (Fig. 6). Thus, 0-acetylation would appear to steri- cally hinder peptidoglycan hydrolysis in a more subtle manner, and it is likely that weak binding of the enzyme along the length of the modified peptidoglycan strands occurs. Molec- ular dynamic simulations of HEWL [27], based on exper- iments concerned with fluorescence anisotropy [28 ] , nuclear magnetic resonance relaxation [29] and nuclear magnetic res-

t \ Y

X U

E

9

> 7 3

2.5,j

2.0 t 1.5 i

l.O!

0.5 f __I

0.0 0 20 40 60 % 0-Acetylat ion

Fig. 6. Dependence of catalytic efficiency for the solubilization of peptidoglycan by H E WL on the degree of 0-acetylation. The steady- state parameters and apparent second-order rate constants were re- calculated after substrate concentrations had been adjusted to account for the putative decrease in effective substrate-binding sites due to the presence of N,U-diacetylmuramyl residues on the various peptido- glycan preparations (see text for details). Closed symbols, native pepti- doglycan; open symbols, chemically de-0-acetylated peptidoglycan preparations. Vmax/Km is expressed as lo3 . A600 min-' . mM-'

onance coupling constants [30], reveal significant structural fluctuations, and this flexibility may allow for the accommo- dation of the protruding 0-acetyl moieties.

Regardless of the mechanism by which 0-acetyl groups inhibit the action of certain muramidases on peptidoglycan, the biological significance of the modification is of great im- portance. The optimal expression of many of the pathobiological activities of peptidoglycan [l l , 13, 31 - 341, including the induction of arthritis, require the persistent cir- culation of large peptidoglycan fragments in the host [ l l , 351. Acute inflammation in the rat model is optimally caused by fragments 5 MDa in size, whereas glycan chains approxi- mately 50 MDa cause chronic erosive joint lesions [35]. Hence, the results reported in this study and by others, on the resist- ance of 0-acetylpeptidoglycan to muramidases, indicate the need for further intensive investigations of the phenomenon.

This research was funded by operating grants to A. J. Clarke from the Natural Science and Engineering Research Council (N. S. E. R. C.), Medical Research Council and The Banting Research Foundation and a postgraduate scholarship to C. Dupont from N. S. E. R. C.

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