Human thymidylate synthase with loop 181-197 stabilized in an inactive conformation: ligand interactions, phosphorylation, and inhibition profiles

Thymidylate synthase (TS) is a well-validated cancer target that undergoes conformational switching between active and inactive states. Two mutant human TS (hTS) proteins are predicted from crystal structures to be stabilized in an inactive conformation to differing extents, with M190K populating the inactive conformation to a greater extent than A191K. Studies of intrinsic fluorescence and circular dichroism revealed that the structures of the mutants differ from those of hTS. Inclusion of the substrate dUMP was without effect on M190K but induced structural changes in A191K that are unique, relative to hTS. The effect of strong stabilization in an inactive conformation on protein phosphorylation by casein kinase 2 (CK2) was investigated. M190K was highly phosphorylated by CK2 relative to an active-stabilized mutant, R163K hTS. dUMP had no detectable effect on phosphorylation of M190K; however, dUMP inhibited phosphorylation of hTS and R163K. Studies of temperature dependence of catalysis revealed that the E(act) and temperature optimum are higher for A191K than hTS. The potency of the active-site inhibitor, raltitrexed, was lower for A191K than hTS. The response of A191K to the allosteric inhibitor, propylene diphosphonate (PDPA) was concentration dependent. Mixed inhibition was observed at low concentrations; at higher concentrations, A191K exhibited nonhyperbolic behavior with respect to dUMP and inhibition of catalysis was reversed by substrate saturation. In summary, inactive-stabilized mutants differ from hTS in thermal stability and response to substrates and PDPA. Importantly, phosphorylation of hTS by CK2 is selective for the inactive conformation, providing the first indication of physiological relevance for conformational switching.     

Activities of anionic and cationic trypsins in the temperature range from 5 to 37 degrees C. Mutant anionic trypsins as a model of cold-adapted (psychrophilic) enzymes

Temperature dependences of kinetic constants (k _cat and K _m) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of coldadapted fishes. The anionic and cationic trypsins were found to differ in organization of the S₁ -substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S₁) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K _m value) to the anionic and cationic trypsins and on the catalytic activity k _cat should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.     

Variants of human thymidylate synthase with loop 181–197 stabilized in the inactive conformation

Loop 181–197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180°. In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k_cat/K_m reduced by factors in a 2–12 range. One of the mutants, M190K, is however unique in having the value of k_cat/K_m smaller by a factor of ～7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181–197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188–194 of this loop, assumes a different conformation with the positions of C_α shifted up to 7.2 Å. This affects region 116–128, which became ordered in M190K while it is disordered in wt. The conformation of 116–128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.     

Evaluation of kinetic data: What the numbers tell us about PRMTs

Protein arginine N-methyltransferase (PRMT) kinetic parameters have been catalogued over the past fifteen years for eight of the nine mammalian enzyme family members. Like the majority of methyltransferases, these enzymes employ the highly ubiquitous cofactor S-adenosyl-l-methionine as a co-substrate to methylate arginine residues in peptidic substrates with an approximately 4-μM median K_M. The median values for PRMT turnover number (k_cat) and catalytic efficiency (k_cat/K_M) are 0.0051 s⁻¹ and 708 M⁻¹ s⁻¹, respectively. When comparing PRMT metrics to entries found in the BRENDA database, we find that while PRMTs exhibit high substrate affinity relative to other enzyme-substrate pairs, PRMTs display largely lower k_cat and k_cat/K_M values. We observe that kinetic parameters for PRMTs and arginine demethylase activity from dual-functioning lysine demethylases are statistically similar, paralleling what the broader enzyme families in which they belong reveal, and adding to the evidence in support of arginine methylation reversibility.     

Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase

Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA_CCCG, which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N^G-monomethyl-l-arginine, l-citrulline and l-homoarginine were incorporated successfully into proteins using this method. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8, R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications.     

STUDIES ON THE KINETICS OF ACETYLCHOLINESTERASE IN THE RESISIANT AND SUSCEPTIBLE STRAINS OF HOUSEFLY (MUSCA DOMESTICA)

Abstract Acetylcholinesterase (AChE) in the susceptible (S) and the resistant (R) strains of housefly (Musca domestica) was investigated using kinetic analysis. The V_max values of AChE for hydrolyzing acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were 4578.50 and 1716.08nmol/min/mg* protein in the R strain, and were 1884.75 and 864.72 nmol/min/mg. protein in the Sstrain, respectively. The V_max ratios of R to S enzyme were 2.43 for ATCh and 1.98 for BTCh. The K_m values of AChE for ATCh and BTCh were 0.069 and 0.034 mmol/L in the S strain, and 0.156, 0.059 mmol/L in the R strain, respectively. The K_m ratios of R to S enzyme were 2.26 for ATCh and 1.74 for BTCh. The k_i ratios of S to R enzyme for three insecticides propoxur, methomyl and paraoxon were 46.04, 4.17 and 2. 86, respectively. In addition, k_cat and k_cat/K_m for measuring turnover and catalytic efficiency of AChE were determined using eserine as titrant. The k_cat values of AChE from the R strain for both ATCh and BTCh were higher than those values from the S strain. But the values of k_cat/K_m were in contrary to the k_cat values with R enzyme compared to S enzyme. The AChE catalytic properties and sensitivity to the inhibition by three insecticides in the R and S strains of housefly were discussed based on contribution of V_max, K_m, k_i, k_cat and k_cat/K_m. All these data implied that AChE from the R strain might be qualitatively altered. We also observed an intriguing phenomenon that inhibitors could enhance the activity of AChE from the resistant strain. This “flight reaction” of the powerful enzyme might be correlated with the developing resistance of housefly to organophosphate or carbamate insecticides.     

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (k_cat/k_m) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono-layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.     

A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications

In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k_cat/K_M)_Glc/(k_cat/K_M)_Gal = 172], the variant oxidizes both sugars equally well [(k_cat/K_M)_Glc/(k_cat/K_M)_Gal = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K_M values and approximately ten-fold higher k_cat values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60°C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D -glucose/D -galactose mixtures at both 30 and 50°C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 Å resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.     

An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the β-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k_cat for carboxylase activity, a 2-fold increase in K_m for CO₂, and a 1.9-fold increase in K_m for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k_cat and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

Use of peptidyl-4-methoxy-2-naphthylamides to assay plasmin

Several tripeptide amides of 4-methoxy-2-naphthylamide were synthesized and tested as possible substrates for human plasmin. These peptides contained arginine or lysine as the carboxyl terminus. One such amide, benzyloxycarbonyl-Ala-Ala-Lys-4-methoxy-2-naphthylamine (CBZ-Ala-Ala-Lys-MNA), was found to be a better substrate for plasmin than for other mammalian serine proteases. Measuring the release of 4-methoxy-2-naphthylamine fluorometrically or by colorimetry after coupling with fast blue B dye provided convenient assays for as little as 0.05 CTA unit of plasmin. K_m and k_cat values obtained were 0.90 m and 0.7 sec⁻¹, respectively. The assays were simple, sensitive, versatile, and specific.     

Structure-function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high-resolution structures

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf‐type (Nii1 and Nii3), and root‐type (Nii2 and Nii4). The high‐resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe–4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3‐M175G, Nii3‐M175E, and Nii3‐M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3‐Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild‐type (Nii3‐WT) and the Met175 variants showed that the hydrophobic side‐chain of Met175 facilitated enzyme efficiency (k_cat/K_m). The Nii4‐WT has Lys449 at the equivalent position of Gln448 in Nii3‐WT. The enzyme activity assay revealed that the turnover number (k_cat) and Michaelis constant (K_m) of Nii4‐WT were lower than those of Nii3‐WT. However, the k_cat/K_m of Nii4‐WT was about 1.4 times higher than that of Nii3‐WT. A comparison of the kinetics of the Nii3‐Q448K and Nii4‐K449Q variants revealed that the change in k_cat/K_m was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low‐affinity and Nii4 is the high‐affinity aNiR.     

The R163K mutant of human thymidylate synthase is stabilized in an active conformation: structural asymmetry and reactivity of cysteine 195

Loop 181-197 of human thymidylate synthase (hTS) populates two conformational states. In the first state, Cys195, a residue crucial for catalytic activity, is in the active site (active conformer); in the other conformation, it is about 10 A away, outside the active site (inactive conformer). We have designed and expressed an hTS variant, R163K, in which the inactive conformation is destabilized. The activity of this mutant is 33% higher than that of wt hTS, suggesting that at least one-third of hTS populates the inactive conformer. Crystal structures of R163K in two different crystal forms, with six and two subunits per asymmetric part of the unit cells, have been determined. All subunits of this mutant are in the active conformation while wt hTS crystallizes as the inactive conformer in similar mother liquors. The structures show differences in the environment of catalytic Cys195, which correlate with Cys195 thiol reactivity, as judged by its oxidation state. Calculations show that the molecular electrostatic potential at Cys195 differs between the subunits of the dimer. One of the dimers is asymmetric with a phosphate ion bound in only one of the subunits. In the absence of the phosphate ion, that is in the inhibitor-free enzyme, the tip of loop 47-53 is about 11 A away from the active site.     

Arginine and lysine as products of basic carboxypeptidase activity associated with fibrinolysis

Blood carboxypeptidases play an important role in the regulation of fibrinolysis. We have proposed here the method for the assay of blood carboxypeptidase activity associated with coagulation/fibrinolysis using the natural substrate fibrin and the detection of basic amino acids arginine and lysine as products under conditions closely resembling those in vivo. Plasma samples from 15 patients with arterial hypertension have been investigated. Coagulation and subsequent fibrinolysis were initiated by addition of standard doses of thrombin and tissue plasminogen activator, respectively. Arginine and lysine concentrations before, during, and after completion of fibrinolysis were determined using HPLC. The parameters of fibrinolysis were evaluated by the clot turbidity assay. The coagulation/fibrinolysis cycle was accompanied by a significant increase in concentrations of arginine and lysine in the incubation mixture by 101 and 81%, respectively. The duration of fibrinolysis initiation significantly correlated with the degree of increase of these amino acids: r _S = −0.733 and −0.761 for arginine and lysine, respectively (p < 0.05). Arginine generation had two maximums: one in the beginning of clot lysis and another one at the end of the lysis, whereas the lysine release occurred mainly in the middle of fibrinolysis. Thus, the carboxypeptidase activity associated with fibrinolysis can be considered as a local source of the essential amino acids originated from fibrin clot degradation products.     

Identification of Residues Surrounding the Active Site of Type A Botulinum Neurotoxin Important for Substrate Recognition and Catalytic Activity

Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain (LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar residues resulted in drastic lowering of the catalytic rate constant (k _cat), but had less effect on substrate affinity (K _m). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k _cat/K _m) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation to Asn resulted in 140-fold decrease in k _cat/K _m. Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163, Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165 with Leu resulted in fourfold increase in k _cat/K _m. For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe, respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC competent for catalysis or provide conformational selection of the substrate.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.     

Semi-rational site-directed mutagenesis of phyI1s from <Emphasis Type="Italic">Aspergillus niger</Emphasis> 113 at two residue to improve its phytase activity

Through alignment of amino acid sequences among different phytases, we found that the amino acid at residues 53 and 91 vary broadly. To prove that the amino acid at residues 53 and 91 were related to phytase specific activity, two single mutant phyI1s Q53R and K91D were obtained by site-directed mutagenesis strategy. None of the single amino acid residues in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants (Q53R and K91D) indicated that the mutants were attributed to 2.2- and 1.5-fold increased specific activity, and a 1.47- and 1.16-fold increased affinity for sodium phytate. In addition, the overall catalytic efficiency (k _cat/K _m) of the two mutants was improved 4.08- and 2.84-fold compared to that of the wild type. Such mutants will be instrumental for the structure–function study of the enzyme and for industrial application.     

Lysine 194 Is Functional in Isocitrate Lyase from Escherichia coli

Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had k_cat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. K _m values for Mg²⁺-D_s-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased K _m value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state. Received: 3 December 1996 / Accepted: 18 December 1996     

Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5–5, RA95.5–8, and RA95.5–8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site‐directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in k _cat relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in k _cat/K _M across the series. Only modest decreases in k _cat/K _M are observed for the E85Q variants with little effect on k _cat. Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst.     

Re‐examining the role of Lys67 in class C β‐lactamase catalysis

Lys67 is essential for the hydrolysis reaction mediated by class C β‐lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C β‐lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2‐ to 3‐fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C β‐lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61‐fold decrease in k_cat, the deacylation rate. We have determined the structure of the K67R mutant by X‐ray crystallography both in apo and transition state‐analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C β‐lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.