The stereochemical pathway of lysozyme-catalyzed glycosidic bond hydrolysis has been shown to proceed with at least 99. Lysozyme-catalyzed glycosidic bond hydrolysis has been shown to be largely carbonium ion due to the kinetic isotope effect of a-deuterium. Thus, the substrate structure for the enzyme in cell walls appears to be a linear polymer of the disaccharide NAG-NAM with all linkages.
CH COOH
The results of these studies indicate that all of these saccharides are bound to the main cleft in the surface of the enzyme. The studies on the NAG complex show that the α and 13 forms of the sugar do not bind in exactly the same way. The authors propose that a mechanism involving help by the acetamido group of the substrates of lysozyme is of potential importance to the enzyme-catalyzed reaction.
PART I
CHAPTER I
This observation provides us with a method for calculating the binding constants for inhibitors and substrates of lysozyme. As an example of the difference spectrum obtained upon interaction of lysozyme and chitotriose at pH 5.3, we can say that two ionizable groups on the enzyme are disrupted by the presence of the trimer sub-.
CHAPTER II
09, which can be unambiguously assigned to the methyl group protons of the acetamido side chain (P. Two resonances instead of one were observed for these acetamidomethyl protons in the presence of the enzyme. The most reasonable explanation for the observed shifts for the acetamido-methyl protons across the bond from acetamido sugars to lyso-.
CHAPTER III
1 It was further demonstrated in that section that in the presence of lysozyme the acetamido-methyl proton resonances of the a.- and fl-anomeric forms of NAG were resolved and also that the two forms around the same binding site(s) on the enzyme. The ratio of the slope of the a-NAG data to that of S-NAG {provided [a. More interesting, however, is the significant difference in the chemical shifts of the bound forms of each anomer (6.
Our previous measurements of the association of a-methyl-NAG and S-methyl-NAG with lysozyme, using p. This small difference combined with the observation that a.- and 13-NAG compete for the same site on the enzyme (Part I, Chapter II) will usually provide good evidence that the binding of the anomers is identical. As a result, the orientation of the acetamido-methyl group has changed somewhat with respect to a tryptophan residue (number 108 in the amino acid sequence) on the enzyme.
Chemical shifts of the acetamido m*:thyl group were measured against the internal acetone standard. This was achieved by multiple ultrafiltrations of a solution of the enzyme in a Diaflo Ultrafiltration. 8 to observe chemical shift differences of nuclei in α-NAG and 8-NAG in the presence of the enzyme, compared to the chemical shifts of the same nuclei in the absence of the enzyme, (Pb)a.
Representation of chemical shift data (from Table 1) for the methyl acetamido resonances of α-NAG and S-NAG (in mutarotation equilibrium) relative to lysozyme.
EQUILIBRIUM
14 MIN
5 MIN
FREE
CHAPTER IV
In this study, both the glycosidic and acetamidomethyl resonances were observed in the presence of the enzyme and the effects were quantified as a function of temperature and pH. 3 (Tracerlab, Inc.) to exchange the acetate impurity in the enzyme preparation which at this pH disrupted the acetamidomethyl resonance of the substrate, and finally lyophilized. The chemical shift of the glycosidic methyl group in the bound state was determined by measuring the ratio of the glycosidic methyl group shift to the acetamidomethyl group shift.
This result does not exclude the possibility of a conformational change in the enzyme leading to weak binding of the glycoside. However, the acetamido-methyl resonance shows two breaks due to ionizations on the enzyme substrate complex that affect the magnetic environment of the acetamido group while it is bound. 7 is interesting in that the ionization of the relevant group has no noticeable influence on binding.
The effect of this group is most likely manifested as an electric field or magnetic anisotropy change that affects the magnetic environment of the binding site. Therefore, this group must be close to the binding site of the acetamido-methyl group of 13-MeNAG. Plot of the temperature dependence of the dissociation constant (K .. 8) for S-methyl-NAG and lysozyme.
The pH dependence of the chemical shifts ( 6 ) of the glycosidic methyl proton - e - and acetamidomethylene.
CHAPTER V
This conclusion was reached by determining the chemical shift induced in the acetamidomethyl protons of the two anomeric forms upon association with the enzyme. The chemical shift of the glycoside methyl group in the bound state was determined by measuring the ratio of the glycosidic methyl group shift to the acetamidomethyl group shift. The deuterated compound lacks the acetamido resonance to a higher field and therefore this resonance in chitobiose can be attributed to the acetamido group on the pyranose ring at the reducing end of the disaccharide.
The chemical shift of the resonance to a higher field closely matches that due to the acetamidomethyl group at the reducing end of chitobiose (see Table 1) and is therefore assigned to the methyl group at the reducing end of chitotriose. In addition, a poorly resolved doublet was observed, as in the case of the trisaccharide. We also studied the association of the methyl glycosides of chitobiose and chitotriose with lysozyme.
We therefore also assign this resonance to the internal acetamido group of methyl-13-chitotrioside and the downfield resonance to the acetamido-methyl group at the non-glycosidic end of the trisaccharide glycoside. Since the o:.- and 13-anomeric forms of the disaccharide were present at mutarotational equilibrium in the sample chitobiose used,. The pH dependence of the magnetic environment of the reducing-end acetamido-methyl group while bound was measured over the pH range 2.
The data obtained for the chitobiose binding site show that there are at least three ionizable groups that affect the environment of the reducing end acetamido-methyl group.
ESH)
This resonance was broadened, but by a smaller amount than that observed for the methyl acetamido resonance at the glycosidic end of the molecule. Calculation of the chemical shift, 6., for the methyl acetamido protons near the glycosidic end of the disaccharide glycoside gave a value of O. The calculated chemical shift of this resonance for the bound form of the companion small molecule was O. The methyl acetamido group distal to the glycosidic end did not undergo any change in chemical shift as a result of binding to lysozyme.
Under these conditions, a broadening of the resonances was observed, and it was also evident that the resonance corresponding to the acetamido methyl group at the reducing end of the trisaccharide underwent a chemical shift to higher field. The resonance corresponding to the acetamido methyl group at the reducing end of the trisaccharide was clearly resolved and was further shifted to higher field. The value of A for the acetamido methyl resonance of the acetamido methyl resonance at the reducing end at the lowest s0/E0 ratio was 0.
The resonance corresponding to the central acetamido group of the trisaccharide did not appear to undergo any chemical shift due to association with lysozyme. It allows discrimination from the magnetic environment associated with the site on the enzyme occupied by the non-reducing terminal acetamidomethyl group of chitotriose. The resonance due to the middle acetamido-m·3thyl group of the glycoside did not appear to undergo any chemical shift change due to macromolecular association, while the acetamido-methyl group did.
This explanation was proposed by Thomas (1968) to account for the large increase in the line width of the reducing end of the acetamido methyl group resonance of chitobiose and the resonance due to the acetarnido methyl group proximal to the glycosidic end of methyl -S-chitobioside in the presence of lysozyme. Furthermore, due to the similarity of the acetamido-methyl group shifts of methyl-[3-chitobioside and chitobiose, it appears that a- and. Plot of the reciprocal of the observed chemical shift (o) for the reducing-end acetamido methyl group resonance of chitobiose versus varying chitobiose concentrations (S0) i.
NAG~NAG- : NAG/
I NAG~R
I NAG,R
CHAPTER VI
An example of the effect of temperature on the absorptions of acetamido methyl groups of chitobiose in the presence of lysozyme at pH 5. The authors derived an expression for the overall magnetization of the system in the xy plane, G (the applied field is in the z direction). The large error in the experimental determination of the chemical shift suggests that the exchange rate is still intermediate.
This trend in linewidth appears to correlate fairly well with the binding strength of subsites A, B, and C. Given the estimated error in determining the rate constants, only one mole of E greater than 2 kcal should be -1 . This suggests that saccharide binding involves a transition state with a very unfavorable entropy of activation.
7 produces a remarkable effect on the activation energy for the formation of the disaccharide enzyme complex. The most reasonable explanation for the increase is that the ionization of a particular group on the enzyme surface creates a potential energy barrier to the formation of the complex. A kind of short-range repulsive force caused by the ionization of the base form of the enzyme.
This seems to provide more information than just the rate constant measurements.
