2Department of Microbiology, 933 19th Street South, 545 CHSB-19, University of Alabama at Birmingham, Birmingham AL 35294, USA, and 3Department of Biochemistry and Molecular Genetics, 933 19th Street South, 545 CHSB-19, University of Alabama at Birmingham, Birmingham AL 35294, USA
Received on August 18, 2000; revised on November 27, 2000; accepted on December 11, 2000.
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Abstract |
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Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of Kms for all of these different enzymes.
Key words: catalytic mechanism/hyaluronan/hyaluronate lyase/kinetics/Streptococcus pneumoniae
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Introduction |
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This bacterial organism presents a variety of proteins on its surface that are often antigenic, and the antibodies against them exhibit protective properties for the host (Paton, 1998; Nabors et al., 2000
). These proteins interact with the host cells, take part in various stages of bacterial colonization or invasion of the host and often are indispensable for pneumococci (Austrian, 1982
; Busse, 1991
). Examples of such protein antigens are pneumococcal surface protein A (McDaniel et al., 1991
; Briles et al., 1997
; Jedrzejas et al., 2000
), choline-binding protein A (Rosenow et al., 1997
), autolysin (Lock et al., 1988
), and hyaluronate lyase (SpnHL) (Berry et al., 1994
; Jedrzejas et al., 1998a
,b; Li et al., 2000
; Ponnuraj and Jedrzejas, 2000). Hyaluronate lyase is an enzyme that is either cell-associated or released outside of the pneumococci. It degrades primarily hyaluronan and certain chondroitin sulfates, major components of the hosts extracellular matrix of tissues (ECM) (Linker et al., 1956
; Boulnois, 1992
; Menzel and Farr, 1998
). This degradative process assists pneumococci in its spread to various host tissues. Also, as the structure of ECM is destroyed, the host cells are exposed to other pneumococcal toxins, such as pneumolysin (Feldman et al., 1992
).
Hyaluronan (HA), a major substrate for hyaluronate lyase, is abundantly present in the ECM of essentially all vertebrates. It is a polymeric substance built from repeating disaccharide unit of hyaluronic acid (Figure 1) (Laurent, 1970). The polymer interacts with water to create a strikingly viscoelastic solution. These unique mechanical properties are utilized in, for example, joints as a shock absorber (Winter and Arnott, 1977
). However, in addition to mechanical properties, hyaluronan synthesis and degradation is finely regulated and hyaluronan is involved in multiple signal transduction processes (Comper and Laurent, 1978
; Fraser and Laurent, 1989
) often utilizing other macromolecules, such as CD44 or RHAM (Yang et al., 1994
; Laurent and Fraser, 1992
).
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Results |
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Initial-velocity experiments with a hexasaccharide hyaluronan substrate
The kinetic parameters of the wild-type and mutant enzymes are interpreted in the context of a mechanism (Figure 4A and B) including (1) a substrate binding step, (2) a catalytic step, (3) a product release step, (4) a translocation step, and (5) proton exchange with microenvironment. The translocation step directly relates to the processive character of the enzyme shown previously (Pritchard et al., 1994). As a consequence of the initial-velocity approximation the product release step is irreversible. Based on this mechanism, Equation 1 for the enzymes initial velocity,
i, was derived by the method of net rate constants (Cleland, 1977
).
|
1
The Vm/Km, the first-order rate constant as the substrate concentration approaches zero, the Vm, the initial velocity as the substrate concentration approaches infinity, and the Km, the Vm divided by Vm/Km, are expressed in terms of the rate, ki, and thermodynamic equilibrium, Ki, constants of the component steps (Equations 2, 3, and 4).
2
3
4
Three patterns of effects of the mutations on the various steps of the enzymatic reaction catalyzed by hyaluronate lyase can be measured by the steady-state methods employed here. The magnitude of each of the measured effects depends on the magnitude of the primary effect on one or more of the steps as well as the extent to which these steps are rate determining for the parameter measured. These effects, described by Equations 2 and 3, are as follows: (1) changes only in Vm/Km associated with various mutant forms indicate that the particular form of the enzyme binds the substrate at a different affinity (K1); (2) changes only in Vm indicate that the mutant enzyme translocates the nascent product/substrate at a different rate (k7); and (3) changes in both Vm/Km and Vm indicate either that the mutation in the enzyme affects both binding and translocation or that the mutant enzyme carries out catalysis, product release, or both at different rates. Although the Km may approximate the dissociation constant of the enzymesubstrate complex under the same circumstances, it can also be affected by many other rate and equilibrium constants in the system.
Polymeric hyaluronan and its hexasaccharide units
The wild-type enzyme obeyed Michaelis-Menten kinetics. The values of Vm and Km for degradation of the hyaluronan hexasaccharide substrate were 463 (±18) mMol/(min * mg) and 0.08 (± 0.02) mM, respectively (Figure 5A, Table I). The SpnHL enzyme catalyzed degradation of human umbilical cord polymeric hyaluronan, HA, by the wild-type enzyme gave Vm and Vm/Km indistinguishable from those for the hexasaccharide substrate (Table I). To compare both sets of results the concentration of polymeric HA was expressed in the form of the concentration of the hexasaccharide units of HA. Further experiments with polymeric HA were unsatisfactory due to the inhomogeneity of available preparations of this substrate which varied substantially in its molecular weight from 3.0 x 106 to 5.8 x 106 Da (Laurent, 1970) and in the presence of significant impurities in the available hyaluronan, such as other forms of glycosaminoglycans. These other glycosaminoglycans could act on the enzyme, for example, as inhibitors and distort the results. Therefore, the experiments with mutated enzyme forms were performed with the hexasaccharide of the substrate.
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The mutation R243V has almost equal effects on both Vm and Vm/Km but only minimal effects on Km (Figure 5, Table I). According to the structural model of the enzyme complex with HA6 (Li et al., 2000), this residue is significantly removed from the catalytic residues. The side chain of Arg243 interacts with the glucuronate carboxyl group of the penultimate disaccharide moiety, the glucosamine N-acetyl group of the third disaccharide moiety, and the glycosidic oxygen of the ß1,4 linkage between them, which is the penultimate glycosidic bond to be cleaved.
The mutant N580G is affected significantly only in the Vm/Km and the Km, suggesting that only the substrate binding is affected (Table I). This result confirms the predictions from the crystal structure (Li et al., 2000) because Asn580 is close enough to the substrate for interactions and it interacts only with the penultimate disaccharide in the hexasaccharide substrate. Specifically, the side chain of Asn580 interacts with the hydroxyl group on C5 of the penultimate N-acetyl-glucosamine residue.
Finally the influence of the Asn580 residue only on the binding of HA is consistent with the structural information. All hypotheses presented in the analysis of wild-type and mutant enzymes kinetics are consistent with and confirm our previous structural and mechanistic studies of the enzyme (Li et al., 2000; Ponnuraj and Jedrzejas, 2000).
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Discussion |
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Kinetic properties of the mutant forms of the enzyme
All three of the catalytic mutants of hyaluronate lyase are expected to reduce the value of both the Vm and the Vm/Km. The N349A mutation removes the amide nitrogen, which otherwise would participate in a hydrogen bond with the carboxyl group of the glucuronic moiety of the nonreducing end of the disaccharide to be removed. Thus, the interaction of the asparagine residue acting as a partial electron sink to assist in the removal of the proton on C5 would be unavailable. The H399A mutation substitutes a more highly electronegative and geometrically different group for the histidine that acts as a base to remove a proton from the C5 of the glucuronyl residue of the leaving disaccharide. In the Y408F mutation, a very poor acid is substituted for a reasonably acidic tyrosine. Therefore, in the mutant enzyme it will be practically impossible for a proton to be donated to the oxygen subtending the scissile bond. The very low activity of this mutant, which required in the excess of hundred times as much enzyme to demonstrate the activity, is possibly due to donation of a proton directly by water molecules in the microenvironment of the enzymes catalytic cleft.
The placement of the positively charged Arg243 residue at the nonreducing end of the cleft suggests that it guides the electronegative substrate in the cleft and aids in the positioning of the substrate for catalysis. This residue forms hydrogen bonds and salt bridges with HA2 and HA3 and therefore plays an important role in stabilizing this segment of the substrate chain in the cleft. The valine, substituted at Arg243, would not be expected to participate in any similar interactions. It is hypothesized that the interactions of this residue with the acetamide of the substrate is important for both translocation and product release. Due to the placement of this residue it seems unlikely that the Arg243 is directly involved in the release of the disaccharide product; it must therefore be involved with the nascent polysaccharide for which product release is a part of translocation. Although there is some ambiguity about the processivity and the role of translocation in the cleavage of the substrate (hexasaccharide) in the present investigation, a cleaved and translocated hexasaccharide (tetrasaccharide) would have either none or altered interactions with R243. The fact that a hydrogen bond at this rather remote location promotes the overall reaction independent of binding alone indicates that this Arg243 is important for translocation and that translocation is important for the cleavage of this substrate.
The mutations of the noncatalytic residues were designed to test the different roles of these residues predominantly in controlling access to the cleft and in binding of the enzyme to the substrate. Asn580 is located in the narrowest part of the cleft (Figure 3A, B) and as such it might regulate the access of the substrate to the enzyme. Also, it is the only residue from the ß-sheet domain of the enzyme, which is in contact with the substrate. Therefore, this mutation could provide some insight into the role of the ß-domain in the catalytic cycle. The N580G mutation would change the position and increase the electronegativity of the group as well as widen the cleft in its narrowest point. This opening up of the cleft could facilitate the binding of the substrate to the enzyme. Based on the model structure of the hexasaccharide and disaccharide product complexes, this residue is known to interact with HA2, and the mutation from Asn to Gly decreases the interaction with the C6 hydroxyl of the N-acetylglucosamine of the penultimate disaccharide, resulting in a lower binding constant and nonproductive binding (Table I).
Comparison with polysaccharide-degrading enzymes utilizing hydrolysis to degrade their substrates
To our knowledge there is no precise data available describing the kinetic properties of polysaccharide lyases in general. However, some data is available for enzymes degrading polysaccharides through a hydrolysis mechanism, such as Bacillus agaradherans endonuclease (Davies et al., 1998) or Asoergillus niger glucoamylase (Christensen et al., 1997
). The hydrolytic degradation for these enzymes likely proceeds for both enzymes through a double-displacement mechanism (Koshland, 1953
) with a net retention of the anomeric configuration of the substrate/product (Davies et al., 1998
). The Km determined for the wild-type B. agaradherans endonuclease with substrates of various lengths range from 0.047 to 0.33 mM with the standard error of
10 % of the value (Davies et al., 1998
). Similarly the Kms of the G1 and G2 wild-type forms of A. niger glucoamylase are 0.18 ± 0.02 mM and 0.28 ± 0.06 mM, respectively (Christensen et al., 1997
). These Kms are comparable with the wild-type S. pneumoniae hyaluronate lyase Km value of 0.14 ± 0.04 mM (Table I) (Jedrzejas, 2000
). Therefore, for both types of polysaccharide-degrading enzyme utilizing different degradation mechanisms, PAD for polysaccharide lyases and double displacement (DD) for hydrolases, the Km values seem to be fairly consistent and are approximately 0.1 mM.
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Materials and methods |
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Initial-velocity measurement and data analysis
The reaction progress curves for the native and mutated forms were obtained as described previously (Jedrzejas et al., 1998a,b) by measurement of the absorbance at 232 nm, due to the unsaturated disaccharide product. The reaction for each enzyme was carried out in 100 µl of 50 mM sodium acetate and 10 mM CaCl2 at pH 6.0 with substrate concentrations ranging from 0.012 mM to 5 mM hexasaccharide, HA6, in the above buffer. The reaction was initiated by the addition of 10 µl of enzyme in the same buffer. The various amounts of enzyme in the reaction mixture were as follows: wild-type (33 ng), R243V (50 ng), N349A (550 ng), H399A (275 ng), Y408F (5410 ng), and N580G (33 ng). The product absorbance was measured every 5 s in a Beckman DU640 spectrophotometer for 1.5 min at 22°C. The reaction progress was linear over the time of measurement within the precision of the instrument. The initial velocity of the reaction was determined from the increase in absorbance over the first 90 s of the reaction. The slope was divided by the molar absorption coefficient for the disaccharide product, 5.5 x 103 mol1 cm1, (Yamagata et al., 1963
).
For the polymeric hyaluronan degradation experiments, the HA concentration in solution was expressed as moles of hexasaccharide based on a hexasaccharide molecular weight of 1203.9 g. The concentrations of enzyme and substrate were identical to those mentioned above for the wild-type enzyme setup.
The data for initial velocity, vi, from each experiment in which the concentration of substrate, S, was varied, were fit to the Michealis-Menten equation, v = VmS/(Km + S), with a nonlinear regression program (Scientist Micromath) where Km is the Michaelis constant and Vm is the maximum velocity. For all experiments, goodness-of-fit statistics showed that R2 and correlation values were greater than 0.988 and 0.981, respectively. Among the curve-fitting results, the program gave values of Vm and Km as well as their respective standard deviations (). From the latter data the values of Vm/Km and the 95% confidence limits (3 x
) were calculated.
Other methods
The enzyme concentration was determined by either the Bradford protein assay (Bradford, 1976) with bovine serum albumin as standards or by the ultraviolet absorption at 280 nm using the molar extinction coefficient calculated based on the SpnHL amino acid sequence data (Pace et al., 1995
; Jedrzejas et al., 1998a
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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