Kinetic properties of Streptococcus pneumoniae hyaluronate lyase

Stephen J. Kelly2, Kenneth B. Taylor3, Songlin Li2 and Mark J. Jedrzejas1,2

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.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment.

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


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Streptococcus pneumoniae is a Gram-positive bacterial pathogen, normally colonizing the nasopharyngeal cavity of humans, causing significant mortality and morbidity worldwide. In addition to causing very serious and often fatal diseases, such as pneumonia, bacteremia, or meningitis, these bacteria cause less dangerous but significantly more prevalent diseases, such as sinusitis or otitis media (Mufson, 1990Go; Boulnois, 1992Go). The groups of human population especially affected by this organism are children who have not yet fully developed immune systems, the elderly, and people with compromised immune systems, such as diabetics or those with acquired immunodeficiency syndrome (Gray et al., 1980Go; Johnston, 1991Go; Musher, 1991Go; Fiore et al., 1999Go).

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, 1998Go; Nabors et al., 2000Go). 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, 1982Go; Busse, 1991Go). Examples of such protein antigens are pneumococcal surface protein A (McDaniel et al., 1991Go; Briles et al., 1997Go; Jedrzejas et al., 2000Go), choline-binding protein A (Rosenow et al., 1997Go), autolysin (Lock et al., 1988Go), and hyaluronate lyase (SpnHL) (Berry et al., 1994Go; Jedrzejas et al., 1998aGo,b; Li et al., 2000Go; 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 host’s extracellular matrix of tissues (ECM) (Linker et al., 1956Go; Boulnois, 1992Go; Menzel and Farr, 1998Go). 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., 1992Go).

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, 1970Go). 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, 1977Go). 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, 1978Go; Fraser and Laurent, 1989Go) often utilizing other macromolecules, such as CD44 or RHAM (Yang et al., 1994Go; Laurent and Fraser, 1992Go).



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Fig. 1. Structure of hyaluronan substrate and the representation of the degradation process catalyzed by hyaluronate lyase. The final degradation product of the pneumococcal enzyme is the unsaturated disaccharide unit of hyaluronan.

 
The enzyme hyaluronate lyase has been cloned, overexpressed, and purified (Berry et al., 1994Go; Jedrzejas et al., 1998aGo), thus, extensive biochemical and biophysical characterization of the enzyme has been possible. The availability of large quantities of the protein led to its crystallization (Jedrzejas et al., 1998bGo) and the three-dimensional structure determination of its native form, together with a model of a complex with the hexasaccharide unit of hyaluronan (Li et al., 2000Go) as well as of a complex with the disaccharide product of hyaluronan degradation (Ponnuraj and Jedrzejas, 2000) (Figures 2 and 3). The analysis of these structures, together with the results of mutation of selected residues involved in catalysis, led to elucidation of the enzymatic mechanism of the pneumococcal hyaluronate lyase and likely hyaluronate lyases from other bacterial organisms, which is termed proton acceptance and donation (PAD) (Li et al., 2000Go). This degradative process is thought to proceed via a processive mode in which the random initial enzyme binding to hyaluronan is followed by degradation of the same substrate chain toward the nonreducing end until the whole substrate is degraded (Pritchard et al., 1994Go). For the pneumococcal hyaluronate lyase and for several other known bacterial hyaluronate lyases, the final degradation product is an unsaturated disaccharide derivative of hyaluronic acid (Jedrzejas et al., 1998aGo). Here we report further analysis of the properties of this enzyme and additional insight into its catalytic mechanism from studies of the kinetic properties of the wild-type and the mutant forms of this lyase.



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Fig. 2. General structure of the S. pneumoniae hyaluronate lyase enzyme (ribbon rendition of the whole enzyme with selected catalytic and substrate binding residues depicted in the ball and stick fashion) with the modeled hexasaccharide unit of hyaluronan (ball and stick rendition) bound in the catalytic cleft of the enzyme. Both structural domain, {alpha} and ß, and the active center (cleft) are labeled. The figure was created with Molscript (Kraulis, 1991) using Brookhaven Protein Data Bank (pdb) coordinates: 1egu.

 



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Fig. 3. (A) Detailed structure of the cleft with the modeled hexasaccharide unit of hyaluronan. The side chains for the three catalytic residues, Asn349, His399, and Tyr408, and the substrate binding residues, Arg243 and Asn580, are drawn. (B) The graphical representation of the side chains of the mutated residues analyzed in this study. The orientation is the same as for the part A of this figure.

 

    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Overexpression and purification of the wild-type and mutant enzymes
The recombinant S. pneumoniae hyaluronate lyase from Escherichia coli used in this study was obtained as previously described (Jedrzejas et al., 1998aGo). Also, the hexasaccharides of hyaluronan were obtained by the digestion of human umbilical cord hyaluronan using Streptomyces hyalurolyticus hyaluronate lyase and confirmed by mass spectrometry experiments as previously described (Shimada and Matsmura, 1980Go; Li et al., 2000Go). The mutant forms of the pneumococcal enzyme were produced using standard cloning techniques as reported elsewhere (Maniatis et al., 1982Go; Li et al., 2000Go). The residues for the mutation studies were selected by the analysis of the three-dimensional structural information available about the pneumococcal hyaluronate lyase that only recently became available (Li et al., 2000Go; Ponnuraj and Jedrzejas, 2000).

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., 1994Go). As a consequence of the initial-velocity approximation the product release step is irreversible. Based on this mechanism, Equation 1 for the enzyme’s initial velocity, {nu}i, was derived by the method of net rate constants (Cleland, 1977Go).




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Fig. 4. (A) Chemical formulation of the kinetic mechanism of S. pneumoniae hyaluronate lyase. Small kis refer to rate constants of individual steps. The step with the forward rate constant, k1, is the substrate binding step; that by k3 is the catalytic step, that by k5 is product release, and that by k7, is a translocation step. The abbreviations used are: E = enzyme, S = hyaluronan substrate, EPD = enzyme with bound truncated hyaluronan substrate and a disaccharide product, EP = enzyme with bound truncated hyaluronan substrate and disaccharide product released from the active site of the enzyme. (B) Proposed catalytic mechanism for the enzyme. Only the catalytic residues, Asn349, His399, Tyr408, and two disaccharide units, HA1 and HA2, are shown. The movement of protons is indicated by arrows.

 

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 enzyme–substrate 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, 1970Go) 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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Fig. 5. (A) Comparison of similar kinetic properties of the wild-type S. pneumoniae hyaluronate lyase enzyme acting on the polymeric hyaluronan, circles, and on its hexasaccharide units squares. (B) Comparison of the different kinetic properties of the wild-type hyaluronate lyase, circles, and the R243V mutant, squares, degrading hexasaccharides of hyaluronan. The insert represents double reciprocal graph of 1/V (V, velocity of the reaction) versus 1/S (S, substrate concentration) for the wild-type and R243V mutant. Kinetic experiments were carried out as described in Materials and methods.

 

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Table I. The values of the steady-state kinetic parameters Vm, Km, and Vm/Km and their standard deviations for the pneumococcal hyaluronate lyase degrading hyaluronan substrate.
 
Kinetic properties of the mutant forms of the enzyme
The availability of the crystal structures of the wild-type pneumococcal hyaluronate lyase (Li et al., 2000Go) and its complex with the disaccharides product (Ponnuraj and Jedrzejas, 2000), as well as the model structure of the hexasaccharide HA in the active cleft of the enzyme, provide a unique opportunity to correlate the kinetic and structural properties of this enzyme. The mutations of residues H399A, N349A, and Y408F, which all take part in the catalytic reaction and all resulting mutated enzymes have significantly reduced values of Vm and Vm/Km as compared to the wild-type enzyme (Figure 5B). The hypothesis that each of these mutations reduces the rate of the catalytic step is supported by these results as well as the structural evidence that these residues all interact with the substrate atoms that participate in catalysis. Of these three, only the H399A mutation increases the value of the Km significantly. Although changes in Km may not be strictly due to alterations in the binding of substrate, such an interpretation in the present case is more credible because of the absence of a significant effect on Km in the other two mutants that produced a substantial effect on the catalytic rate. Based on the structural information available, the side chain oxygen of Tyr408 interacts through a hydrogen bond with the glycosidic oxygen of the ß1,4 linkage between the disaccharide units at the nonreducing end of the HA1 disaccharide (Figure 3A). The NE2 nitrogen atom of His399 is pointing toward and interacts with the C5 carbon of the first glucuronate at the nonreducing end. The latter interaction seems important for binding as well as for catalysis. The side chain of Asn349 is in direct contact with the carboxyl group of the same glucuronate moiety, the oxygen atom in the glycosidic linkage, and the N-acetyl group of the glucosamine of the penultimate disaccharide. It is interesting to speculate that the His399 seems to exert its effect more during binding, whereas the N349 seems to exert its effect more during catalysis. The Tyr408 residue forms a hydrogen bond with the glycosidic oxygen atom of the scissile bond (Figure 4B).

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., 2000Go), 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., 2000Go) 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., 2000Go; Ponnuraj and Jedrzejas, 2000).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Structural properties of the enzyme
The crystal structure of S. pneumoniae hyaluronate lyase shows that the enzyme is built at least from two domains, a catalytic domain composed predominantly of {alpha}-helices and a supportive one built predominantly from ß-sheets (Jedrzejas et al., 1998aGo,b; Li et al., 2000Go). The {alpha}-helical domain encompasses an elongated cleft in the middle of the molecule. This cleft region has only minimal interactions with the ß-sheet domain (Figures 2, 3A, B). The negatively charged substrate binds to the predominantly positively charged cleft. The positive charges in the cleft originate from lysines and arginines highly conserved residues in different species. The size of the cleft limits the number to three disaccharide units of the substrate that can interact with the enzyme. Thus, a hexasaccharide unit of hyaluronan is the largest substrate size interacting in the cleft. These three disaccharide units can only fit in the cleft in one orientation to interact properly with the catalytic residues of the enzyme. These units are numbered from the reducing end to the nonreducing end of the hexasaccharide substrate as HA1, HA2, and HA3 (Figure 3A). The active site is located at one side of the cleft and is composed of two parts, an aromatic patch and a catalytic group, each composed of three residues (Figure 3A,B). The aromatic patch is built from the three aromatic residues Trp291, Trp292, and Phe343, and according to the earlier structural and mechanistic studies this patch is responsible for the selection of cleavage sites on the substrate chain. The catalytic group, which is composed of His399, Tyr408, and Asn349, is responsible for the cleavage of the glycosidic ß1,4 linkage between HA1 and HA2 chains of the substrate through a five-step PAD mechanism (Li et al., 2000Go). The side chain of catalytic Asn349 forms several hydrogen bonds with the carboxyl group of HA1 glucuronate moiety and acts as a partial electron sink for the carboxyl group of the glucuronic acid of the disaccharide to be cleaved. These hydrogen bonds attract the electronegative charge of the carboxyl group away from the C5 carbon of the glucuronate group, leading to acidification of the C5 hydrogen. This hydrogen (proton) then becomes withdrawn by His399, which acts as a base. The loss of this proton results in the rehybridization of C5. At the same time, Tyr408 acts as an acid and donates one proton to the glycosidic oxygen, which leads to the cleavage of its covalent bond with HA1. C4 is rehybridized and a C4–C5 double bond is formed. The enzyme likely loses the extra proton on His399 to the surrounding water molecules and attracts one proton to Tyr408, in this way returning to the original state, ready for the next round of catalysis (Figure 4B). The unsaturated disaccharide product is released, and the hyaluronan substrate is translocated so that the penultimate disaccharide then interacts with the three catalytic residues (Figure 4).

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 enzyme’s 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., 1998Go) or Asoergillus niger glucoamylase (Christensen et al., 1997Go). The hydrolytic degradation for these enzymes likely proceeds for both enzymes through a double-displacement mechanism (Koshland, 1953Go) with a net retention of the anomeric configuration of the substrate/product (Davies et al., 1998Go). 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., 1998Go). 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., 1997Go). These Kms are comparable with the wild-type S. pneumoniae hyaluronate lyase Km value of 0.14 ± 0.04 mM (Table I) (Jedrzejas, 2000Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Hyaluronan used in this study was from human umbilical cord and was purchased from Sigma Chemical (sodium salt, Lot # 53H0439). All other chemicals were purchased either from Fisher Scientific or Sigma Chemical. The hexasaccharide units of hyaluronan were obtained by digesting the human umbilical cord hyaluronan using the S. hyalurolyticus hyaluronate lyase (Shimada and Matsmura, 1980Go) as previously described (Li et al., 2000Go). The identity and high purity of the hexasaccharide substrates was confirmed using mass spectrometry experiments. The recombinant S. pneumoniae hyaluronate lyase enzyme from E. coli and its mutants R243V, N349A, H399A, Y408F, and N580V used in this study were also obtained as previously described (Jedrzejas et al., 1998aGo,b; Li et al., 2000Go). The activity unit for the enzyme was defined as the molar amount of the enzyme that produces 1 µM of the product per min (Jedrzejas et al., 1998aGo). The native and mutant enzymes were stored in 10 mM Tris–HCl, pH 7.4, 2 mM EDTA, and 1 mM DTT buffer at 5 mg/ml protein concentration at –80°C until the kinetic experiments were performed. Under such conditions the enzyme loss of activity with storage was minimized.

Initial-velocity measurement and data analysis
The reaction progress curves for the native and mutated forms were obtained as described previously (Jedrzejas et al., 1998aGo,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 mol–1 cm–1, (Yamagata et al., 1963Go).

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 ({sigma}). From the latter data the values of Vm/Km and the 95% confidence limits (3 x {sigma}) were calculated.

Other methods
The enzyme concentration was determined by either the Bradford protein assay (Bradford, 1976Go) 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., 1995Go; Jedrzejas et al., 1998aGo).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Ms. Ejvis Lamani for her help and assistance. This work was supported by a grant from the National Institutes of Health AI 44078 (to M.J.J.).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
DD, double displacement; DTT, dithiothreitol; ECM, extracellular matrix of tissues; EDTA, disodium ethylenediamine tetraacetate; HA, polymeric hyaluronan; HA6, hexasaccharide units of hyaluronan; PAD, proton acceptance and donation; SpnHL, Streptococcus pneumoniae hyaluronate lyase.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Austrian, R. (1982) Some observations on the pneumococcus and on the current status of pneumococcal disease and its prevention. In: Quie, P.G., and Kass, E.H. (eds.) The Pneumococcus and the pneumocpccal vaccine. University of Chicago Press, Chicago, pp. 191–207.

Berry, A.M., Lock, R.A., Thomas, S.M., Rajan, D.P., Hansman, D., and Paton, J.C. (1994) Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli. Infect. Immun., 62, 1101–1108.[Abstract]

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Feldman, C., Munro, N.C., Jeffery, P.K., Mitchell, T.J., Andrew, P.W., Boulnois, G.J., Guerreiro, D., Rohde, J.A., Todd, H.C., and Cole, P.J. (1992) Pneumolysin induces the salient histologic features of pneumococcal infection in the rat lung in vivo. Am. J. Respir. Cell Mol. Biol., 5, 416–423.[ISI]

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