Engineered dimer interface mutants of triosephosphate isomerase: the role of inter-subunit interactions in enzyme function and stability

The role of inter-subunit interactions in maintaining optimal catalytic activity in triosephosphate isomerase (TIM) has been probed, using the Plasmodium falciparum enzyme as a model. Examination of subunit interface contacts in the crystal structures suggests that residue 75 (Thr, conserved) and residue 13 (Cys, variable) make the largest number of inter-subunit contacts. The mutants Cys13Asp (C13D) and Cys13Glu (C13E) have been constructed and display significant reduction in catalytic activity when compared with wild-type (WT) enzyme (～ 7.4-fold decrease in k(cat) for the C13D and ～ 3.3-fold for the C13E mutants). Analytical gel filtration demonstrates that the C13D mutant dissociates at concentrations <1.25 μM, whereas the WT and the C13E enzymes retain the dimeric structure. The order of stability of the mutants in the presence of chemical denaturants, like urea and guanidium chloride, is WT > Cys13Glu > Cys13Asp. Irreversible thermal precipitation temperatures follow the same order as well. Modeling studies establish that the Cys13Asp mutation is likely to cause a significantly greater structural perturbation than Cys13Glu. Analysis of sequence and structural data for TIMs from diverse sources suggests that residues 13 and 82 form a pair of proximal sites, in which a limited number of residue pairs may be accommodated.     

Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing-β-1, 3-glucanase

Laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of β-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.     

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI

Three mutants of Escherichia coli ribonuclease HI, in whichan invariant acidic residue Asp134 was replaced, were crystallized,and their three-dimensional structures were determined by X-raycrystallography. The D134A mutant is completely inactive, whereasthe other two mutants, D134H and D134N, retain 59 and 90% activitiesrelative to the wild-type, respectively. The overall structuresof these three mutant proteins are identical with that of thewild-type enzyme, except for local conformational changes ofthe flexible loops. The ribonuclease H family has a common activesite, which is composed of four invariant acidic residues (Asp10,G1u48, Asp70 and Asp134 in E.coli ribonuclease HI), and theirrelative positions in the mutants, even including the side-chainatoms, are almost the same as those in the wild-type. The positionsof the -polar atoms at residue 134 in the wild-type, as wellas D134H and D134N, coincide well with each other. They arelocated near the imidazole side chain of His124, which is assumedto participate in the catalytic reaction, in addition to thefour invariant acidic residues. Combined with the pH profilesof the enzymatic activities of the two other mutants, H124Aand H124A/D134N, the crystallographic results allow us to proposea new catalytic mechanism of ribonuclease H, which includesthe roles for Asp134 and His124.     

Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH

Xylanase C from Aspergillus kawachii has an optimum pH of 2.0 and is stable at pH 1.0. The crystal structure of xylanase C was determined at 2.0 A resolution (R-factor = 19.4%). The overall structure was similar to those of other family 11 xylanases. Asp37 and an acid-base catalyst, Glu170, are located at a hydrogen-bonding distance (2.8 A), as in other xylanases with low pH optima. Asp37 of xylanase C was replaced with asparagine and other residues by site-directed mutagenesis. Analyses of the wild-type and mutant enzymes showed that Asp37 is important for high enzyme activity at low pH. In the case of the asparagine mutant, the optimum pH shifted to 5.0 and the maximum specific activity decreased to about 15% of that of the wild-type enzyme. On structural comparison with xylanases with higher pH optima, another striking feature of the xylanase C structure was found; the enzyme has numerous acidic residues concentrated on the surface (so-called 'Ser/Thr surface' in most family 11 xylanases). The relationship of the stability against extreme pH conditions and high salt concentrations with the spacially biased distribution of charged residues on the proteins is discussed.      

Modification of S1 subsite specificity in the cysteine protease cathepsin B

Cysteine proteases of the papain family generally exhibit broadP₁ specificity. A notable exception is papaya proteinase IV(PPIV), which only accepts Gly at this position. In all othercysteine proteases the S₁ subsite residues 23 and 65 (papainnumbering) are absolutely conserved as Gly, while in PPIV theyare replaced by Glu and Arg, respectively. These differencesappear to underlie both PPIV specificity and its resistanceto inhibition by cystatins. To test this hypothesis, the equivalentresidues (Gly27 and Gly73) in the mammalian cysteine proteasecathepsin B were changed to Glu and Arg, respectively. Relativeto the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showeda drastic reduction in activity with substrates containing aP₁ Arg. In contrast, substrates having a Gly residue in P₁ werehydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg)exhibited no detectable activity against any substrate studied.Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane]was found to be similar to that of the wild-type enzyme. Incontrast, inhibition by cystatin C exhibited a 20 000-fold reduction.These results demonstrate the dramatic influence of side chainsat sequence locations 27 and 73 on the S₁ subsite specificityof cysteine proteases.     

Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.     

Characterization of BlsM, a nucleotide hydrolase involved in cytosine production for the biosynthesis of blasticidin S

Biosynthesis of the antifungal agent blasticidin S in Streptomyces griseochromogenes requires the formation of free cytosine. The blsM gene in the blasticidin S gene cluster is predicted to encode a protein that has sequence homology with several nucleoside transferases. In vitro analysis of recombinant BlsM revealed that the enzyme functions as a nucleotide hydrolase and catalyzes the formation of free cytosine by using cytidine 5'-monophosphate (CMP) as the preferred substrate. Cytosine production was significantly lower with CDP, CTP, and dCMP as alternate substrates. BlsM was also observed to have low-level cytidine deaminase activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Point mutations were introduced in blsM at putative catalytic residues to generate three mutant enzymes, BlsM Ser98Asp, Glu104Ala, and Glu104Asp. All three mutants lost CMP hydrolysis activity, but the Ser98Asp mutant showed a modest increase in cytidine deaminase activity.     

Identification of four amino acid residues essential for catalysis in human cytidine deaminase by site-directed mutagenesis and chemical modifications

By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild- type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.      

Mutational analyses of restriction endonuclease--HindIII mutant E86K with higher activity and altered specificity

We have performed mutational analyses of restriction endonucleaseHindIII in order to identify the amino acid residues responsiblefor enzyme activity. Four of the seven HindIII mutants, whichhad His-tag sequences at the N-termini, were expressed in Escherichiacoli, and purified to homogeneity. The His-tag sequence didnot affect enzyme activity, whereas it hindered binding of theDNA probe in gel retardation assays. A mutant E86K in whichLys was substituted for Glu at residue 86 exhibited high endonucleaseactivity. Gel retardation assays showed high affinity of thismutant to the DNA probe. Surprisingly, in the presence of atransition metal, Mo²⁺ or Mn²⁺, the E86K mutant cleaved substrateDNA at a site other than HindIII. Substitution of Glu for Valat residue 106 (V106E), and Asn for Lys at residue 125 (K125N)resulted in a decrease in both endonucleolytic and DNA bindingactivities of the enzyme. Furthermore, substitution of Leu forAsp at residue 108 (D108L) abolished both HindIII endonucleaseand DNA binding activities. CD spectra of the wild type andthe two mutants, E86K and D108L, were similar to each other,suggesting that there was little change in conformation as aresult of the mutations. These results account for the notionthat Asp108 could be directly involved in HindIII catalyticfunction, and that the substitution at residue 86 may bringabout new interactions between DNA and cations.     

Elucidation of the role of Ser329 and the C-terminal region in the catalytic activity of Pseudomonas stutzeri L-rhamnose isomerase

Pseudomonas stutzeri l-rhamnose isomerase (l-RhI) is capable of catalyzing the isomerization between various aldoses and ketoses, showing high catalytic activity with broad substrate-specificity compared with Escherichia coli l-RhI. In a previous study, the crystal structure of P. stutzeri l-RhI revealed an active site comparable with that of E. coli l-RhI and d-xylose isomerases (d-XIs) with structurally conserved amino acids, but also with a different residue seemingly responsible for the specificity of P. stutzeri l-RhI, though the residue itself does not interact with the bound substrate. This residue, Ser329, corresponds to Phe336 in E. coli l-RhI and Lys294 in Actinoplanes missouriensis d-XI. To elucidate the role of Ser329 in P. stutzeri l-RhI, we constructed mutants, S329F (E. coli l-RhI type), S329K (A. missouriensis d-XI type), S329L and S329A. Analyses of the catalytic activity and crystal structure of the mutants revealed a hydroxyl group of Ser329 to be crucial for catalytic activity via interaction with a water molecule. In addition, in complexes with substrate, the mutants S329F and S329L exhibited significant electron density in the C-terminal region not observed in the wild-type P. stutzeri l-RhI. The C-terminal region of P. stutzeri l-RhI has flexibility and shows a flip-flop movement at the inter-molecular surface of the dimeric form.     

Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

Knowledge of protein stability principles provides a means toincrease protein stability in a rational way. Here we explorethe feasibility of stabilizing proteins by replacing solvent-exposedhydrogen-bonded charged Asp or Glu residues by the neutral isostericAsn or Gln. The rationale behind this is a previous observationthat, in some cases, neutral hydrogen bonds may be more stablethat charged ones. We identified, in the apoflavodoxin fromAnabaena PCC 7119, three surface-exposed aspartate or glutamateresidues involved in hydrogen bonding with a single partnerand we mutated them to asparagine or glutamine, respectively.The effect of the mutations on apoflavodoxin stability was measuredby both urea and temperature denaturation. We observed thatthe three mutant proteins are more stable than wild-type (onaverage 0.43 kcal/mol from urea denaturation and 2.8°C froma two-state analysis of fluorescence thermal unfolding data).At high ionic strength, where potential electrostatic repulsionsin the acidic apoflavodoxin should be masked, the three mutantsare similarly more stable (on average 0.46 kcal/mol). To ruleout further that the stabilization observed is due to removalof electrostatic repulsions in apoflavodoxin upon mutation,we analysed three control mutants and showed that, when thecharged residue mutated to a neutral one is not hydrogen bonded,there is no general stabilizing effect. Replacing hydrogen-bondedcharged Asp or Glu residues by Asn or Gln, respectively, couldbe a straightforward strategy to increase protein stability.     

Connecting Active‐Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop‐6 (residues 166–176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98 % of the 6277 unique TIM sequences), spatially proximal to E165 and the loop‐6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue—phenylalanine—at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop‐6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms.     

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions

The role of electrostatic interactions between the ionizableAsp158 and the active site thiolate-imidazolium ion pair ofsome cysteine proteinases has been the subject of controversyfor some time. This study reports the expression of wild typeprocaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants fromEscherichia coli. Purification of autocatalytically maturedenzymes yielded sufficient fully active material for pH (k_cat/K_m)profiles to be obtained. Use of both uncharged and charged substratesallowed the effects of different reactive enzyme species tobe separated from the complications of electrostatic effectsbetween enzyme and substrate. At least three ionizations aredetectable in the acid limb of wild type caricain and the Gluand Asn mutants. Only two pK_a, values, however, are detectablein the acid limb using the Ala mutant. Comparison of pH activityprofiles shows that whilst an ionizable residue at position158 is not essential for the formation of the thiolate-imidazoliumion pair, it does form a substantial part of the electrostaticfield responsible for increased catalytic competence. Changingthe position of this ionizable group in any way reduces activity.Complete removal of the charged group reduces catalytic competenceeven further. This work indicates that hydronations distantto the active site are contributing to the electrostatic effectsleading to multiple active ionization states of the enzyme.     

Improving the thermostability of Bacillus stearothermophilus neutral protease by introducing proline into the active site helix

A proline residue was introduced into the N-terminus (Ile140 and Asp141), the middle (Leu147) and the C-terminus (Asp153) of the active site helix of Bacillus stearothermophilus neutral protease for comparing the effects on the thermostability. Introduction of a proline residue into the N-terminus at sites 140 and 141 increased the half- survival temperature (HST) by 7.5 and 2.8 degrees C, respectively, from 68.3 degrees C of the wild-type enzyme. A proline residue at Leu147 decreased the HST by 10.2 degrees C, while no change was observed by introducing a proline residue in the C-terminus. These results were coincidental with the CD data which indicated increases in Tm values of 4.4 and 2.3 degrees C for I140P and D141P, respectively. Susceptibility to alpha-chymotrypsin hydrolysis markedly decreased in mutants I140P and D141P, while increasing in L147P. Molecular modeling suggested that glycine residues on the N-terminus side of proline residues in I140P and D141P relaxed the possible strain caused by proline introduction. The thermostability can, therefore, be explained based on changes in the molecular rigidity.      

Roles of catalytic residues in {alpha}-amylases as evidenced by the structures of the product-complexed mutants of a maltotetraose-forming amylase

The crystal structures of the four product-complexed singlemutants of the catalytic residues of Pseudomonas stutzeri maltotetraose-forming-amylase, E219G, D193N, D193G and D294N, have been determined.Possible roles of the catalytic residues Glu219, Asp193 andAsp294 have been discussed by comparing the structures amongthe previously determined complexed mutant E219Q and the presentmutant enzymes. The results suggested that Asp193 predominantlyworks as the base catalyst (nucleophile), whose side chain atomlies in close proximity to the C1-atom of Glc4, being involvedin the intermediate formation in the hydrolysis reaction. WhileAsp294 works for tightly binding the substrate to give a twistedand a deformed conformation of the glucose ring at position–1 (Glc4). The hydrogen bond between the side chain atomof Glu219 and the O1-atom of Glc4, that implies the possibilityof interaction via hydrogen, consistently present throughoutthese analyses, supports the generally accepted role of thisresidue as the acid catalyst (proton donor).     

Altered ionization of the B13 Glu in insulin B9 and B10 mutants: a computational analysis

An experimentally determined pK(a) change of +2.50 units has been reported for the B13 Glu residue in a dimeric B9 Ser --> Asp insulin mutant relative to the native dimer. Poisson-Boltzmann electrostatics-based pK(a) calculations were performed to probe the effect of the B9 Ser --> Asp and B10 His --> Asp mutations on aggregation and the ionization behaviour of the B13 carboxylate. The method produced shifts of +2.64 and +2.45 units for the pK(a) shift of the two B13 residues in the B9 mutant dimer relative to the wild-type dimer, which is in good agreement with the experimental value (<6% error). The calculations also suggest that the reason neither mutant insulin can aggregate into hexamers is the resultant crowding of negatively charged groups in the central solvent channel on hexamer formation. In the wild-type insulin, binding of zinc ions by B10 His overcomes this problem, whereas in the B10 mutant this possibility is ruled out by the absence of the zinc binding site. A series of mutations are predicted to stabilize the medically relevant, monomeric form of insulin.     

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli

When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.      

Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases

Picornavirus 3C proteinases (3C^pro) are cysteine proteinasesbut recent sequence analyses have shown that they are relatedto trypsin-like serine proteinases. Two models of 3C^pro structurehave been presented. Both models indicate that residues His40and Cysl47 are members of the catalytic triad but the modelsdiffer in the designation of the third member of the catalytictriad, which is assigned as either Glu71 or Asp85. To test theimportance of these four residues in the catalytic activityof 3C^pro of coxsackievirus B3, a member of the enterovirus subgroupof the picornavirus family, single amino acid substitutionswere introduced at each of the four sites. All of these mutationsresulted in the reduction or inactivation of autocatalytic cleavageof the 3C precursor protein expressed in Escherichia coli, suggestingthat all of these residues are essential for the proteolyticreaction. The substitution of Cysl47 with Ala abolished 3C^proactivity while the mutant in which Cysl47 was replaced withSer retained reduced proteolytic activity both in cis and intrans. Our results strongly support the proposal that Cysl47of 3C^pro functions as a nucleophile analogous to Serl95 of trypsin-likeserine proteinases.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase

Site-directed mutagenesis was used to alter active-site residuesof methylamine dehydrogenase (MADH) from Paracoccus denitrificans.Four residues of the ß subunit of MADH which are inclose proximity to the tryptophan tryptophylquinone (TTQ) prostheticgroup were modified. The crystal structure of MADH reveals thateach of these residues participates in hydrogen bonding interactionswith other active-site residues, TTQ or water. Relatively conservativemutations which removed the potentially reactive oxygens onthe side chains of Thr122, Tyr119, Asp76 and Asp32 each resultedin greatly reduced or undetectable levels of MADH production.The reduction of MADH levels was determined by assays of activityand Western blots of crude extracts with antisera specific forthe MADH ß subunit. No activity or cross-reactiveprotein was detected in extracts of cells expressing D76N, T122Aand T122C MADH mutants. Very low levels of active MADH wereproduced by cells expressing D32N, Y119F, Y119E and Y119K MADHmutants. The Y119F and D32N mutants were purified from cellextracts and found to be significantly less stable than wild-typeMADH. Only the T122S MADH mutant was produced at near wild-typelevels. Possible roles for these amino acid residues in stabilizingunusual structural features of the MADH ß subunit,protein folding and TTQ biosynthesis are discussed.