Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis.

Aldose-ketose isomerization by xylose isomerase requires bivalent cations such as Mg2+, Mn2+, or Co2+. The active site of the enzyme from Actinoplanes missouriensis contains two metal ions that are involved in substrate binding and in catalyzing a hydride shift between the C1 and C2 substrate atoms. Glu 186 is a conserved residue located near the active site but not in contact with the substrate and not with a metal ligand. The E186D and E186Q mutant enzymes were prepared. Both are active, and their metal specificity is different from that of the wild type. The E186Q enzyme is most active with Mn2+ and has a drastically shifted pH optimum. The X-ray analysis of E186Q was performed in the presence of xylose and either Mn2+ or Mg2+. The Mn2+ structure is essentially identical to that of the wild type. In the presence of Mg2+, the carboxylate group of residue Asp 255, which is part of metal site 2 and a metal ligand, turns toward Gln 186 and hydrogen bonds to its side-chain amide. Mg2+ is not bound at metal site 2, explaining the low activity of the mutant with this cation. Movements of Asp 255 also occur in the wild-type enzyme. We propose that they play a role in the O1 to O2 proton relay accompanying the hydride shift.     

Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine

The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 A resolution using molecular replacement techniques. The genetically engineered molecule catalyses the activation of tyrosine with kinetic properties similar to those of the wild-type enzyme but no longer binds tRNATyr. It contains 319 residues corresponding to the region of the polypeptide chain for which interpretable electron density is present in crystals of the wild-type enzyme. The partly refined model of the wild-type enzyme was used as a starting point in determining the structure of the truncated mutant. The new crystals are of space group P2(1) and contain the molecular dimer within the asymmetric unit. The refined model has a crystallographic R-factor of 18.7% for all reflections between 8 and 2.5 A. Each subunit contains two structural domains: the alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet and the alpha-helical domain (residues 248 to 319) containing five helices. The alpha/beta domains are related by a non-crystallographic dyad while the alpha-helical domains are in slightly different orientations in the two subunits. The tyrosine substrate binds in a slot at the bottom of a deep active site cleft in the middle of the alpha/beta domain. It is surrounded by polar side-chains and water molecules that are involved in an intricate hydrogen bonding network. Both the alpha-amino and hydroxyl groups of the substrate make good hydrogen bonds with the protein. The amino group forms hydrogen bonds with Tyr169-OH, Asp78-OD1 and Gln173-OE1. The phenolic hydroxyl group forms hydrogen bonds with Asp76-OD1 and Tyr34-OH. In contrast, the substrate carboxyl group makes no direct interactions with the enzyme. The results of both substrate inhibition studies and site-directed mutagenesis experiments have been examined in the light of the refined structure.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Zn2+ regulation of ornithine transcarbamoylase. II. Metal binding site

Two types of conformational changes are mediated in Escherichia coli ornithine transcarbamoylase by the metal ion zinc. Upon binding of zinc in rapid equilibrium, the enzyme undergoes an allosteric transition. In the absence of substrates, the zinc-bound enzyme further undergoes a slow isomerization with a concomitant activity loss. Three metal ions are tightly complexed in the isomerized enzyme as determined by gel chromatography and atomic absorption spectroscopy. Since the enzyme is a trimer composed of identical subunits, one zinc ion is bound per enzyme monomer. With the application of site-directed mutagenesis, the cysteinyl residue at position 273 of the enzyme has been identified as a metal ligand. When this residue is replaced by an alanine, zinc is no longer a tight-binding inhibitor and does not promote isomerization. The alteration in the action of zinc on the mutant enzyme is attributed to a reduced metal affinity. The mutant enzyme, when saturated by the metal, displays an intrinsic allostery unchanged from that of the wild-type; an identical Hill coefficient of 1.5 is found for zinc binding to the Ala273 and wild-type enzymes. Cys273 is also a binding site of L-ornithine. At pH 8.5, the Ala273 enzyme binds the substrate analog L-norvaline ten times more weakly and exhibits a kcat/Kmorn that is 27 times less than that of the wild-type enzyme. This finding supports our earlier interpretation that the zinc-induced ornithine co-operativity of ornithine transcarbamoylase is caused by direct competition between L-ornithine and the metal for the same site. As controls, each of the remaining three cysteinyl residues of the bacterial ornithine transcarbamoylase has also been replaced with alanine. These sulfhydryl groups are found not to be related to zinc complexation, ornithine binding or enzyme allostery.     

A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase

Using site-directed mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine. In the closely homologous eukaryotic lactate dehydrogenase, this residue binds the carboxylate group of the substrate by forming a planar bifurcated bond. The mutation diminishes the binding energy of pyruvate, alpha-ketobutyrate and alpha-ketovalerate (measured by kcat/Km) by the same amount (about 6 kcal/mol). For each additional methylene group on the substrate, there is a loss of about 1.5 kcal/mol of binding energy in both mutant and wild-type enzymes. From these parallel trends in the two forms of enzyme, we infer that the mode of productive substrate binding is identical in each, the only difference being the loss of a strong carboxylate-guanidinium interaction in the mutant. In contrast to this simple pattern in kcat/Km, the Km alone increases with substrate-size in the wild-type enzyme, but decreases in the mutant. These results can be most simply explained by the occurrence of relatively tight unproductive enzyme-substrate complexes in the mutant enzyme as the substrate alkyl chain is extended. This does not occur in the wild-type enzyme, because the strong orienting effect of Arg-171 maximizes the frequency of substrates binding in the correct alignment.     

Mechanistic aspects of cyanogenesis from active-site mutant Ser80Ala of hydroxynitrile lyase from Manihot esculenta in complex with acetone cyanohydrin

The structure and function of hydroxynitrile lyase from Manihot esculenta (MeHNL) have been analyzed by X-ray crystallography and site-directed mutagenesis. The crystal structure of the MeHNL-S80A mutant enzyme has been refined to an R-factor of 18.0% against diffraction data to 2.1-A resolution. The three-dimensional structure of the MeHNL-S80A-acetone cyanohydrin complex was determined at 2.2-A resolution and refined to an R-factor of 18.7%. Thr11 and Cys81 involved in substrate binding have been substituted by Ala in site-directed mutagenesis. The kinetic measurements of these mutant enzymes are presented. Combined with structural data, the results support a mechanism for cyanogenesis in which His236 as a general base abstracts a proton from Ser80, thereby allowing proton transfer from the hydroxyl group of acetone cyanohydrin to Ser80. The His236 imidazolium cation then facilitates the leaving of the nitrile group by proton donating.     

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex.

Molecular recognition and site-directed mutagenesis are used in combination to identify kinetically, transition state interactions between glucoamylase (GA) and the substrate maltose. Earlier studies of mutant Glu180----Gln GA had indicated a role in substrate binding for Glu180 (Sierks, M.R., Ford, C., Reilly, P.J. and Svensson, B. (1990) Protein Engng, 3, 193-198). Here, changes in activation energies calculated from measured kcat/Km values for a series of deoxygenated maltose analogues indicate hydrogen bonding between the mutant enzyme and the 3-OH group of the reducing end sugar ring. Using the same substrate analogues and determining activation energies with wild-type GA an additional hydrogen bond with the 2-OH group of maltose is attributed to an interaction with the carboxylate Glu180. This novel combination of molecular recognition and site-directed mutagenesis enables an enzyme substrate transition state contact to be identified and characterized even without access to the three dimensional structure of the enzyme. Given the distant structural relationships between glucoamylases and several starch hydrolases (Svensson,B. (1988) FEBS Lett., 230, 72-76), such identified contacts may ultimately guide tailoring of the activity of these related enzymes.     

Crystal Structures of β-1,4-Galactosyltransferase 7 Enzyme Reveal Conformational Changes and Substrate Binding

The β-1,4-galactosyltransferase 7 (β4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human β4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type β4GalT7 and D211N β4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N β4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an S_N2 type catalytic mechanism for the β4GalT7 enzyme.     

Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme.     

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD⁺ to NADP⁺. In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.     

Random mutagenesis to enhance the activity of subtilisin in organic solvents: Characterization of Q103R subtilisin E

Q103R subtilisin E was isolated following random mutagenesis and screening for improved activity in the presence of dimethylformamide (DMF). Our goal is to identify the mechanism(s) by which amino acid substitutions can enhance enzyme activity in polar organic solvents. A quantitative framework for comparing substrate binding and catalytic activities of mutant and wild-type enzymes in the presence and absence of DMF is outlined. Kinetic experiments performed at high salt concentration (1M KCl) reveal that the mechanism behind the Q103R variant's enhanced activity toward succinyl-Ala-Ala-Pro-Phe-p-nitroanilide is both electrostatic and nonelectrostatic in origin. Favorable electrostatic interactions between the negatively charged succinyl group of the substrate and the positive charge on Arg 103 are responsible for tighter substrate binding. This conclusion is supported by kinetic experiments performed on the related substrate Ala-Ala-Pro-Phe-p-nitroanilide and the hydrolysis kinetics of the Q103E, Q103K, and Q103S variants constructed by site-directed mutagenesis. These results highlight the importance of the choice of the substrate used to screen for improvements in catalytic activity.     

Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase. Elucidation of the roles of amino acids involved in substrate binding and catalysis.

The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm:HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques.     

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala.

The crystal structure of a cytochrome P-450CAM site-directed mutant in which the active site Thr252 has been replaced with an Ala (Thr252Ala) has been refined to an R factor of 0.18 at 2.2 A. According to sequence alignments (Nelson & Strobel, 1989), Thr252 is highly conserved among P-450 enzymes. The crystallographic structure of ferrous camphor- and carbon monoxide-bound P-450CAM (Raag & Poulos, 1989b) suggests that Thr252 is a key active site residue, forming part of the dioxygen-binding site. Mutation of the active site threonine to alanine produces an enzyme in which substrate hydroxylation is uncoupled from electron transfer. Specifically, hydrogen peroxide and "excess" water are produced instead of the product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a local distortion in the distal helix between Gly248 and Thr252 becomes even more severe in the Thr252Ala mutant. Furthermore, a solvent molecule not present in the native enzyme is positioned in the dioxygen-binding region of the mutant enzyme active site. In this location, the solvent molecule could sterically interfere with and destabilize dioxygen binding. In addition, the active site solvent molecule is connected, via a network of hydrogen bonds, with an internal solvent channel which links distal helix residues to a buried Glu side chain. Thus, solvent protons appear to be much more accessible to dioxygen in the mutant than in the wild-type enzyme, a factor which may promote hydrogen peroxide and/or water production instead of substrate hydroxylation. On the basis of crystallographic and mutagenesis data, a proton delivery pathway involving residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type P-450CAM. Coordinates of structures discussed in this paper have been submitted to the Brookhaven Protein Data Bank (Bernstein et al., 1977).     

Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease

Sites for Cys substitutions to form a disulfide bond were chosen in subtilisin E from Bacillus subtilis, a cysteine-free bacterial serine protease, based on the structure of aqualysin I of Thermus aquaticus YT-1 (a thermophilic subtilisin-type protease containing two disulfide bonds). Cys residues were introduced at positions 61 (wild-type, Gly) and 98 (Ser) in subtilisin E by site-directed mutagenesis. The Cys-61/Cys-98 mutant subtilisin appeared to form a disulfide bond spontaneously in the expression system used and showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. The thermodynamic characteristics of these enzymes were examined in terms of enzyme autolysis (t1/2) and thermal stability (Tm). The half-life of the Cys-61/Cys-98 mutant was found to be 2-3 times longer than that of the wild-type enzyme. Similar results were obtained by differential scanning calorimetry. The disulfide mutant showed a Tm of 63.0 degrees C, which was 4.5 degrees C higher than that observed for the wild-type enzyme. Under reducing conditions, however, the characteristics of the mutant enzyme were found to revert to those of the wild-type enzyme. These results strongly suggest that the introduction of a disulfide bond by site-directed mutagenesis enhanced the thermostability of subtilisin E without changing the catalytic efficiency of the enzyme.     

Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase

Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts. To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis. The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme. The apparent affinity of the variant for NADPH was decreased 5–16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained unchanged. This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-fold decrease in K_m for xylose, but only small changes were observed in K_m values for glyceraldehyde. The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5′-phosphate. Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and d-xylose binding in the P. stipitis xylose reductase.     

Functional analysis of point mutations in human flap endonuclease-1 active site.

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.     

Amino acid substitutions in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that influence catalytic activity of the holoenzyme.

Four unique amino acid substitutions were introduced by site-directed mutagenesis into the third conserved region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans (Synechococcus sp., PCC6301), resulting in the formation of four mutant enzymes, I87V, R88K, G91V, and F92L. Wild-type and mutant proteins were purified after synthesis in Escherichia coli. These single amino acid substitutions do not appear to perturb intersubunit interactions or induce any gross conformational changes; purified mutant proteins are stable, for the most part like the wild-type holoenzyme, and exhibit nearly identical CD spectra. Three of the four mutants, however, are severely deficient in carboxylase activity, with kcat less than or equal to 35% of the wild-type enzyme. While the substrate specificity factors were the same for the mutant and wild-type enzymes, significant alterations in some kinetic parameters were observed, particularly in the Michaelis constants for CO2, O2, and RuBP. All four mutant proteins exhibited lower KCO2 values, ranging from 37 to 88% of the wild-type enzyme. Two of the mutants, in addition, exhibited significantly lower KRuBP values, and one mutant showed a substantial decrease in KO2. The effects of the single-site mutations in rbcS of this study strengthen the hypothesis that small subunits may not contribute directly to substrate specificity; however, individual residues of the small subunit substantially influence catalysis by large subunits.     

Interactions of the acid and base catalysts on staphylococcal nuclease as studied in a double mutant.

The mechanism of the phosphodiesterase reaction catalyzed by staphylococcal nuclease is believed to involve concerted general acid-base catalysis by Arg-87 and Glu-43. The mutual interactions of Arg-87 and Glu-43 were investigated by comparing kinetic and thermodynamic properties of the single mutant enzymes E43S (Glu-43 to Ser) and R87G (Arg-87 to Gly) with those of the double mutant, E43S + R87G, in which both the basic and acidic functions have been inactivated. Denaturation studies with guanidinium chloride, CD, and 600-MHz 1D and 2D proton NMR spectra, indicate all enzyme forms to be predominantly folded in absence of the denaturant and reveal small antagonistic effects of the E43S and R87G mutations on the stability and structure of the wild-type enzyme. The free energies of binding of the divalent cation activator Ca2+, the inhibitor Mn2+, and the substrate analogue 3',5'-pdTp show simple additive effects of the two mutations in the double mutant, indicating that Arg-87 and Glu-43 act independently to facilitate the binding of divalent cations and of 3',5'-pdTP by the wild-type enzyme. The free energies of binding of the substrate, 5'-pdTdA, both in binary E-S and in active ternary E-Ca(2+)-S complexes, show synergistic effects of the two mutations, suggesting that Arg-87 and Glu-43 interact anticooperatively in binding the substrate, possibly straining the substrate by 1.6 kcal/mol in the wild-type enzyme. The large free energy barriers to Vmax introduced by the R87G mutation (delta G1 = 6.5 kcal/mol) and by the E43S mutation (delta G2 = 5.0 kcal/mol) are partially additive in the double mutant (delta G1+2 = 8.1 kcal/mol). These partially additive effects on Vmax are most simply explained by a cooperative component to transition state binding by Arg-87 and Glu-43 of -3.4 kcal/mol. The combination of anticooperative, cooperative, and noncooperative effects of Arg-87 and Glu-43 together lower the kinetic barrier to catalysis by 8.1 kcal/mol.     

Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity.

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis. When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type. The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.