Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity, Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 →Ser) and R166K (Arg 166 →Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity,Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 &#77 Ser) and R166K (Arg 166 &#77 Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Relocation of the catalytic carboxylate group in class A beta-lactamase: the structure and function of the mutant enzyme Glu166-->Gln:Asn170-->Asp.

The hydrolysis of beta-lactam antibiotics by the serine-beta-lactamases proceeds via an acyl-enzyme intermediate. In the class A enzymes, a key catalytic residue, Glu166, activates a water molecule for nucleophilic attack on the acyl-enzyme intermediate. The active site architecture raises the possibility that the location of the catalytic carboxylate group may be shifted while still maintaining close proximity to the hydrolytic water molecule. A double mutant of the Staphylococcus aureus PC1 beta-lactamase, E166Q:N170D, was produced, with the carboxylate group shifted to position 170 of the polypeptide chain. A mutant protein, E166Q, without a carboxylate group and with abolished deacylation, was produced as a control. The kinetics of the two mutant proteins have been analyzed and the crystal structure of the double mutant protein has been determined. The kinetic data confirmed that deacylation was restored in E166Q:N170D beta-lactamase, albeit not to the level of the wild-type enzyme. In addition, the kinetics of the double mutant enzyme follows progressive inactivation, characterized by initial fast rates and final slower rates. The addition of ammonium sulfate increases the size of the initial burst, consistent with stabilization of the active form of the enzyme by salt. The crystal structure reveals that the overall fold of the E166Q:N170D enzyme is similar to that of native beta-lactamase. However, high crystallographic temperature factors are associated with the ohm-loop region and some of the side chains, including Asp170, are partially or completely disordered. The structure provides a rationale for the progressive inactivation of the Asp170-containing mutant, suggesting that the flexible ohm-loop may be readily perturbed by the substrate such that Asp170's carboxylate group is not always poised to facilitate hydrolysis.     

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.     

Lysine-73 is involved in the acylation and deacylation of beta-lactamase

Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K(m)), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K(m)) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K(m) vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K(m) at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the acylation and deacylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.     

Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase

The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.     

Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation.

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein-protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild-type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10-70-fold higher with the mutants than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.     

Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis

The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.     

The conserved R166 residue of ALDH5A (succinic semialdehyde dehydrogenase) has multiple functional roles

ALDH5 (aka succinic semialdehyde dehydrogenase) is a NAD(+)-dependent aldehyde dehydrogenase crucial for the proper removal of the GABA metabolite succinic semialdehyde (SSA). All known ALDH5 family members contain the conserved amino acid sequence "MITRK". Our studies of rat ALDH5A indicate that residue R166 in this sequence may play a role in the substrate specificity of ALDH5A for the gamma-carboxylated succinic semialdehyde versus other aliphatic and aromatic aldehydes including acetaldehyde and benzaldehyde. We tested the hypothesis that the R166 residue regulates aldehyde specificity by utilizing rat ALDH5A wild-type (R166wt) and R166K, R166H, R166A, and R166E mutants. The V(MAX) using SSA fell whereas the K(M) for SSA increased for all mutants analyzed yielding k(cat)/K(M) (s(-1)/microM) ratios of 52.3 (R166wt), 5.5 (R166K), 0.01 (R166H), 0.008 (R166E), and 0.004 (R166A). Utilization of acetaldehyde by the R166H mutant was similar to R166wt with k(cat)/K(M)'s of 0.003 and 0.002, respectively. Almost no activity towards acetaldehyde was noted for the R166E and R166A mutants. Unexpectedly, the K(M) for NAD(+) changed: 21 microM (R166wt), 81 microM (R166K), 63 microM (R166H), 35 microM (R166E) and 44 microM (R166A). As release of NADH can be a rate-limiting step for ALDH activity, NADH binding was evaluated for R166wt and R166H enzymes. The K(D) of NADH for R166H (0.9 microM) was 11-fold less than that of ALDH5A wt (10.3 microM) and possibly explains the increase in the K(M) for NAD(+). Furthermore, data using R166K and R166H mutants demonstrate that inhibition of enzyme activity by low pH is regulated in part by the R166 residue. Our data indicate that the R166 residue of ALDH5A regulates multiple enzymatic functions.     

Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1

Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.     

Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region.

Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation. Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity. A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate. To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' [Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223]. The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency. A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu. Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant. These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.     

Differential effects of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and Pr-to-Pfr photoconversion.

Site-directed mutagenesis was performed with the chromophore-bearing N-terminal domain of oat phytochrome A apoprotein (amino acid residues 1-595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore-binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore-binding and the Pr-->Pfr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild-type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild-type protein was found. The light-induced Pr-->Pfr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I700 decay and accelerated Pfr formation. P318 is also involved in the Pr-->Pfr conversion, the millisecond steps (formation of Pfr) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the Pr-->Pfr conversion with kinetics similar to wild-type protein.     

Kinetics of beta-casein hydrolysis by wild-type and engineered trypsin

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in beta-casein were determined by the analysis of kinetics of accumulation of 17 major peptide components revealed by high performance liquid chromatography. When studying pH influence on Arg/Lys bond cleavage preference, averaged rate constants over several Arg&bond;X and Lys&bond;X bonds were used for analysis of kinetics of wild-type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pK(a1) value of 6.5 was found for all studied trypsins. For wild-type trypsin and its K188D/D189K mutant, pK(a2) was found to be 10. The lowest among studied engineered trypsins pK(a2) = 9.3 was determined for K188Y mutant. Considerable preference for the cleavage of Arg over Lys containing peptide bonds was demonstrated for all trypsins with engineered S2 site except for K188H and K188F. The comparison of individual rate constants for various bonds showed that during the hydrolysis by wild-type trypsin, the probabilities of splitting depend on secondary specificity and local hydrophobicity of amino acid residues, which are nearest to the hydrolyzed peptide bond (P2 site). The improvement of prediction of hydrolysis rates performed by the used program was achieved after considering the presence of hydrophobic neighborhood of Lys48--Ile49 and Arg202--Gly203 bonds.     

Two consecutive aspartic acid residues conferring herbicide resistance in tobacco acetohydroxy acid synthase

Acetohydroxy acid synthase (AHAS) catalyzes the first common step in the biosynthesis pathway of the branch chain amino acids in plants and microorganisms. A great deal of interest has been focused on AHAS since it was identified as the target of several classes of potent herbicides. In an effort to produce a mutant usable in the development of an herbicide-resistant transgenic plant, two consecutive aspartic acid residues, which are very likely positioned next to the enzyme-bound herbicide sulfonylurea as the homologous residues in AHAS from yeast, were selected for this study. Four single-point mutants and two double mutants were constructed, and designated D374A, D374E, D375A, D375E, D374A/D375A, and D374E/D375E. All mutants were active, but the D374A mutant exhibited substrate inhibition at high concentrations. The D374E mutant also evidenced a profound reduction with regard to catalytic efficiency. The mutation of D375A increased the K(m) value for pyruvate nearly 10-fold. In contrast, the D375E mutant reduced this value by more than 3-fold. The double mutants exhibited synergistic reduction in catalytic efficiencies. All mutants constructed in this study proved to be strongly resistant to the herbicide sulfonylurea Londax. The double mutants and the mutants with the D375 residue were also strongly cross-resistant to the herbicide triazolopyrimidine TP. However, only the D374A mutant proved to be strongly resistant to imidazolinone Cadre. The data presented here indicate that the two residues, D374 and D375, are located at a common binding site for the herbicides sulfonylurea and triazolopyrimidine. D375E may be a valuable mutant for the development of herbicide-resistant transgenic plants.     

Kinetic characterization of the unisite catalytic pathway of seven beta-subunit mutant F1-ATPases from Escherichia coli

We have studied the kinetics of "unisite" ATP hydrolysis and synthesis in seven mutant Escherichia coli F1-ATPase enzymes. The seven mutations are distributed over a 105-residue segment of the catalytic nucleotide-binding domain in beta-subunit and are: G142S, K155Q, K155E, E181Q, E192Q, M209I, and R246C. We report forward and reverse rate constants and equilibrium constants in all seven mutant enzymes for the four steps of unisite kinetics, namely (i) ATP binding/release, (ii) ATP hydrolysis/synthesis, (iii) Pi release/binding, and (iv) ADP release/binding. The seven mutant enzymes displayed a wide range of deviations from normal in both rate and equilibrium constants, with no discernible common pattern. Notably, steep reductions in Kd ATP were seen in some cases, the value of Kd Pi was high, and K2 (ATP hydrolysis/synthesis) was relatively unaffected. Significantly, when the data from the seven mutations were combined with previous data from two other E. coli F1-beta-subunit mutations (D242N, D242V), normal E. coli F1, soluble and membranous mitochondrial F1, it was found that linear free energy relationships obtained for both ATP binding/release (log k+1 versus log K1) and ADP binding/release (log k-4 versus log K-4). Two conclusions follow. 1) The seven mutations studied here cause subtle changes in interactions between the catalytic nucleotide-binding domain and substrate ATP or product ADP. 2) The mitochondrial, normal E. coli, and nine total beta-subunit mutant enzymes represent a continuum in which subtle structural differences in the catalytic site resulted in changes in binding energy; therefore insights into the nature of energy coupling during ATP hydrolysis and synthesis by F1-ATPase may be ascertained by detailed studies of this group of enzymes.     

Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.     

Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants.

Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady-state and transient-state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt-bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second-order rate constants for compound I reduction (k2app), and decreased first-order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron-transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.     

Mechanistic analyses of catalysis in human pancreatic alpha-amylase: detailed kinetic and structural studies of mutants of three conserved carboxylic acids

The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.     

Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop

A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue.     

Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 beta-lactamase hydrolysing expanded-spectrum cephalosporins.

The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), is characterized by a substrate profile similar to that conferred by these latter enzymes. The role of residues Ala164, His170, Ala171, Asn179, Arg220, Thr237 and Lys242, found in PER-1, was assessed by site-directed mutagenesis. Replacement of Ala164 by Arg yielded an enzyme with no detectable beta-lactamase activity. Two other mutants, N179D and A164R+N179D, were also inactive. Conversely, a mutant with the A171E substitution displayed a substrate profile very similar to that of the wild-type enzyme. Moreover, the replacement of Ala171 by Glu in the A164R enzyme yielded a double mutant which was active, suggesting that Glu171 could compensate for the deleterious effect of Arg164 in the A164R+A171E enzyme. A specific increase in kcat for cefotaxime was observed with H170N, whereas R220L and T237A displayed a specific decrease in activity towards the same drug and a general increase in affinity towards cephalosporins. Finally, the K242E mutant displayed a kinetic behaviour very similar to that of PER-1. Based on three-dimensional models generated by homology modelling and molecular dynamics, these results suggest novel structure-activity relationships in PER-1, when compared with those previously described for the TEM-type ESBLs.