Hydroxyl groups in the betabeta sandwich of metallo-beta-lactamases favor enzyme activity: Tyr218 and Ser262 pull down the lid

Metallo-beta-lactamases (MBLs) efficiently hydrolyze and thereby inactivate various beta-lactam antibiotics in clinical use. Their potential to evolve into more efficient enzymes threatens public health. Recently, we have identified the designed F218Y mutant of IMP-1 as an enzyme with superior catalytic efficiency compared to the wild-type. Thus, it may be found in clinical isolates in the future. In an effort to elucidate the molecular mechanisms involved in enhanced activity, we carried out molecular dynamics simulations of ten MBL variants in complex with a cefotaxime intermediate. The stability of these near-transition state enzyme-substrate intermediate complexes was modeled and compared to the experimental catalytic efficiencies k(cat)/K(M). For each of the ten complexes ten independent simulations were performed. In each simulation the temperature was gradually increased and determined upon breakdown of the complex. Rankings based on the experimental catalytic efficiencies and the data from computer simulations were in good agreement. From trajectory analysis of stable simulations, the combination of Tyr218 and Ser262 was found to lead to an altered hydrogen bonding network, which translates into a closing down movement of a beta-hairpin loop covering the active site. These observations may explain the significantly decreased K(M) and increased k(cat)/K(M) values of this variant toward all substrates recently tested in experiment. Previously, we have discovered that mutations G262S (yielding IMP-1) and G262A in IMP-6 stabilize the Zn(II) ligand His263 and thus the enzyme-substrate intermediate complex through a domino effect, which enhances conversion of drugs like ceftazidime, penicillins, and imipenem. Together, the domino effect and the altered beta-hairpin loop conformation explain how IMP-6 can evolve through mutations G262S and F218Y into an enzyme with up to one order of magnitude increased catalytic efficiencies toward these important antibiotics. Furthermore, the previously proposed binding of a third zinc ion close to the active site of IMP-6 mutant S121G was corroborated by our simulations.     

Impact of remote mutations on metallo-beta-lactamase substrate specificity: implications for the evolution of antibiotic resistance

Metallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.     

A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.     

15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism

Nitrogen-15 NMR spectroscopy has been used to study the hydrogen-bonding interactions involving the histidyl residue in the catalytic triad of alpha-lytic protease in the resting enzyme and in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The 15N shifts indicate that a strong hydrogen bond links the active site histidine and serine residues in the resting enzyme in solution. This result is at odds with interpretations of the X-ray diffraction data of alpha-lytic protease and of other serine proteases, which indicate that the serine and histidine residues are too far apart and not properly aligned for the formation of a hydrogen bond. In addition, the nitrogen-15 shifts demonstrate that protonation of the histidine imidazole ring at low pH in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate triggers the disruption of the aspartate-histidine hydrogen bond. These results suggest a catalytic mechanism involving directed movement of the imidazole ring of the active site histidyl residue.     

Probing the role of Asp-120(81) of metallo-beta-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography

Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.     

Analysis of the context dependent sequence requirements of active site residues in the metallo-beta-lactamase IMP-1

The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.     

The serpin inhibitory mechanism is critically dependent on the length of the reactive center loop

The recent crystallographic structure of a serpin-protease complex revealed that protease inactivation results from a disruption of the catalytic site architecture caused by the displacement of the catalytic serine. We hypothesize that inhibition depends on the length of the N-terminal portion of the reactive center loop, to which the active serine is covalently attached. To test this, alpha(1)-antitrypsin Pittsburgh variants were prepared with lengthened and shortened reactive center loops. The rates of inhibition of factor Xa and of complex dissociation were measured. The addition of one residue reduced the stability of the complex more than 200,000-fold, and the addition of two residues reduced it by more than 1,000,000-fold, whereas the deletion of one or two residues lowered the efficiency of inhibition and increased the stability of the complex (2-fold). The deletion of more than two residues completely converted the serpin into a substrate. Similar results were obtained for the alpha(1)-antitrypsin variants with thrombin and for PAI-1 and PAI-2 with their common target tissue plasminogen activator. We conclude that the length of the serpin reactive center loop is critical for its mechanism of inhibition and is precisely regulated to balance the efficiency of inhibition and stability of the final complex.     

Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations

Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc-containing IMP-1 metallo-beta-lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal-containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc-zinc distance increased by approximately 1 A in the substrate complex compared with the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum-mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc-containing metallo-beta-lactamases as free proteins, in complex with inhibitors and in complex with substrates.     

Engineering d-amino acid containing novel protease inhibitors using catalytic site architecture

The mechanism of proteolysis by serine proteases is a reasonably well-understood process. Typically, a histidine residue acting as a general base deprotonates the catalytic serine residue and the hydrolytic water molecule. We disclose here, the use of an unnatural d-amino acid as a strategic residue in P1 position, designed de novo based on the architecture of the protease catalytic site to impede the catalytic histidine residue at the stage of acyl-enzyme intermediate. Several probe molecules containing d-homoserine or its derivatives at P1 position are evaluated. Compounds 1, 6, and 8-10 produced up to 57% loss of activity against chymotrypsin. More potent and specific inhibitors could be designed with structure optimization as this strategy is completely general and can be used to design inhibitors against any serine or cysteine protease.     

Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants

Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 μs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.     

Chemical mechanism of the serine acetyltransferase from Haemophilus influenzae

The pH dependence of kinetic parameters was determined in both reaction directions to obtain information about the acid-base chemical mechanism of serine acetyltransferase from Haemophilus influenzae (HiSAT). The maximum rates in both reaction directions, as well as the V/K(serine) and V/K(OAS), decrease at low pH, exhibiting a pK of approximately 7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V(1)/E(t), V(1)/K(serine)E(t), V/K(AcCoA)E(t), V(2)/E(t), V(2)/K(OAS)E(t), and V/K(CoA)E(t) are 3300 +/- 180 s(-1), (9.6 +/- 0.4) x 10(5) M(-1) s(-1), 3.3 x 10(6) M(-1) s(-1), 420 +/- 50 s(-1), (2.1 +/- 0.5) x 10(4) M(-1) s(-1), and (4.2 +/- 0.7) x 10(5) M(-1) s(-1), respectively. The K(i) values for the competitive inhibitors glycine and l-cysteine are pH-independent. The solvent deuterium kinetic isotope effects on V and V/K in the direction of serine acetylation are 1.9 +/- 0.2 and 2.5 +/- 0.4, respectively, and the proton inventories are linear for both parameters. Data are consistent with a single proton in flight in the rate-limiting transition state. A general base catalytic mechanism is proposed for the serine acetyltransferase. Once acetyl-CoA and l-serine are bound, an enzymic general base accepts a proton from the l-serine side chain hydroxyl as it undergoes a nucleophilic attack on the carbonyl of acetyl-CoA. The same enzyme residue then functions as a general acid, donating a proton to the sulfur atom of CoASH as the tetrahedral intermediate collapses, generating the products OAS and CoASH. The rate-limiting step in the reaction at limiting l-serine levels is likely formation of the tetrahedral intermediate between serine and acetyl-CoA.     

Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-beta-lactamase

IMP-1 metallo-beta-lactamase (class B) is a plasmid-borne zinc metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics, including carbapenems, rendering them ineffective. Because IMP-1 has been found in several clinically important carbapenem-resistant pathogens, there is a need for inhibitors of this enzyme that could protect broad spectrum antibiotics such as imipenem from hydrolysis and thus extend their utility. We have identified a series of 2,3-(S,S)-disubstituted succinic acids that are potent inhibitors of IMP-1. Determination of high resolution crystal structures and molecular modeling of succinic acid inhibitor complexes with IMP-1 has allowed an understanding of the potency, stereochemistry, and structure-activity relationships of these inhibitors.     

Dominant Intermediate Charcot-Marie-Tooth disorder is not due to a catalytic defect in tyrosyl-tRNA synthetase

Charcot-Marie-Tooth disorder (CMT) is the most common inherited peripheral neuropathy, afflicting 1 in every 2500 Americans. One form of this disease, Dominant Intermediate Charcot-Marie-Tooth disorder type C (DI-CMTC), is due to mutation of the gene encoding the cytoplasmic tyrosyl-tRNA synthetase (TyrRS). Three different TyrRS variants have been found to give rise to DI-CMTC: replacing glycine at position 41 by arginine (G41R), replacing glutamic acid at position 196 by lysine (E196K), and deleting amino acids 153-156 (Δ(153-156)). To test the hypothesis that DI-CMTC is due to a defect in the ability of tyrosyl-tRNA synthetase to catalyze the aminoacylation of tRNA(Tyr), we have expressed each of these variants as recombinant proteins and used single turnover kinetics to characterize their abilities to catalyze the activation of tyrosine and its subsequent transfer to the 3' end of tRNA(Tyr). Two of the variants, G41R and Δ(153-156), display a substantial decrease in their ability to bind tyrosine (>100-fold). In contrast, the E196K substitution does not significantly affect the kinetics for formation of the tyrosyl-adenylate intermediate and actually increases the rate at which the tyrosyl moiety is transferred to tRNA(Tyr). The observation that the E196K substitution does not decrease the rate of catalysis indicates that DI-CMTC is not due to a catalytic defect in tyrosyl-tRNA synthetase.     

Horseradish peroxidase. XXXVI. On the difference between peroxidase and metmyoglobin.

A mechanism for the reaction of hydrogen peroxide with horseradish peroxidase is proposed which involves the catalytic activity of the carboxylate side chain of aspartate residue 43. The corresponding residue in the active site of metmyoglobin is glycine E8, which explains the inability of metmyoglobin to form compound I. Certain aspects of the proposed peroxidase mechanism may be relevant to the catalytic triad for the serine proteases.     

Identification of residues critical for metallo-β-lactamase function by codon randomization and selection

IMP-1 beta-lactamase is a zinc metallo-enzyme encoded by the transferable bla(IMP-1) gene, which confers resistance to virtually all beta-lactam antibiotics including carbapenems. To understand how IMP-1 recognizes and hydrolyzes beta-lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the bla(IMP-1) gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP-1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP-1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild-type enzyme.     

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.     

Mechanism of antigenic drift in influenza virus. Amino acid sequence changes in an antigenically active region of Hong Kong (H3N2) influenza virus hemagglutinin

During antigenic drift in influenza viruses, changes in antigenicity are associated with changes in amino acid sequence of the large hemagglutinin polypeptide, HA1. In ten variants of Hong Kong (H3N2) influenza virus selected with monoclonal antibodies, the proline residue at position 143 in HA1 changed to serine, threonine, leucine or histidine. In other variants, asparagine 133 changed to lysine, glycine 144 to aspartic acid and serine 145 to lysine. All these changes are possible by single base changes in the RNA except the last, which requires a double base change. Residues 142 to 146 also changed in field strains of Hong Kong influenza isolated between 1968 and 1977 (Laver et al., 1980). The single amino acid sequence changes in HA1 of the monoclonal variants were detected by comparing the compositions of the soluble tryptic peptides from the variants with the known sequences of these peptides from wild-type virus. Two insoluble tryptic peptides, comprising residues 110 to 140 and 230 to 255 in the HA1 molecule, were not examined and we do not know if additional changes occurred in these regions.In order to determine whether sequential changes at the same position occurred during antigenic drift, antibody prepared against the new antigenic site on the variants in which proline 143 changed to histidine or threonine was used to select second generation variants of these variants. In the first case, the glycine residue (144) next to the histidine changed to aspartic acid, and in the second, the threonine residue at position 143 reverted to proline and the virus regained the antigenicity of wild-type.Although monoclonal antibodies revealed dramatic antigenic differences between the variants and wild-type virus, only those variants with changes at position 144 of glycine to aspartic acid or at position 145 of serine to lysine could be distinguished from wild-type virus using heterogeneous rabbit or ferret antisera. The other variants, including those which showed sequence changes in widely separated positions of HA1, could not be distinguished from wild-type with heterogeneous antisera.These findings suggest that sequence changes in the region comprising residues 142 to 146 of HA1 affect an important antigenic site on the hemagglutinin molecule, but how these changes affect the antigenic properties, or whether this region actually forms part of the antigenic site is not known.     

Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.     

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.     

The role of the site 342 in catalytic efficiency and pH optima of endoglucanase II from Trichoderma reesei as probed by saturation mutagenesis

The role of site 342 of endoglucanase II from Trichoderma reesei in catalytic efficiency and pH optima was investigated by site saturation mutagenesis. The mutations identified in this study can be divided into three separate classes according to their amino acid features. When Asn342 was substituted by hydrophobic and non-polar amino acids, most variants exhibited an up-shift in pH optimum and their catalytic efficiency was similar to that of the wild-type at their optimal pH. N342R variant had a pH optimum at 6.2. N342K variant did not give an up-shift in pH optimum, although K and R are both amino acids carrying positive charges. Molecular modelling indicated that residue 342 was located at the C-terminus of one of the α-helices near two catalytic residues. Hydrophobic side chains and more H-bonds would make the helix more rigid, which might affect the stability and activity of the enzyme at higher pH.