Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin

We have previously shown that replacing the P1-site residue(Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lysresults in the acquisition of inhibitory activity toward chymotrypsinor trypsin, respectively. However, the inhibitory activitiesthus induced are not strong. In the present study, we introducedadditional amino acid replacements around the reactive siteto try to make the P1-site mutants more effective inhibitorsof chymotrypsin or trypsin. The amino acid replacement AspTyrat the P2' site of OMCHI3(P1Met) resulted in conversion to a35000-fold more effective inhibitor of chymotrypsin with aninhibitor constant (K_i) of 1.17x10^–11 M. The K_i valueof OMCHI3(P1Met, P2'Ala) indicated that the effect on the interactionwith chymotrypsin of removing a negative charge from the P2'site was greater than that of introducing an aromatic ring.Similarly, enhanced inhibition of trypsin was observed whenthe AspTyr replacement was introduced into the P2' site of OMCHI3(P1Lys).Two additional replacements, AspAla at the P4 site and ArgAlaat the P3' site, made the mutant a more effective inhibitorof trypsin with a K_i value of 1.44x10^–9 M. By contrast,ArgAla replacement at the P3' site of OMCHI3(P1Met, P2'Tyr)resulted in a greatly reduced inhibition of chymotrypsin, andAspAla replacement at the P4 site produced only a small changewhen compared with a natural variant of OMCHI3. These resultsclearly indicate that not only the P1-site residue but alsothe characteristics, particularly the electrostatic properties,of the amino acid residues around the reactive site of the proteaseinhibitor determine the strength of its interactions with proteases.Furthermore, amino acids with different characteristics arerequired around the reactive site for strong inhibition of chymotrypsinand trypsin.     

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceousprotease inhibitor which inhibits serine proteases by forminga stable Michaelis complex. The flexible loop region (Thr64–Val69)is a very flexible region in an SSI molecule and its importancein interactions with proteases has been suggested, since conformationalchange of this loop was found to occur for the smooth bindingof SSI with various proteases. In this study, mutated SSIs lackingone or two residues in this region were generated and the effectsof deletions on the interaction with proteases were investigated.Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73)both known to be trypsin inhibitors, to examine the effectsof deletion on interactions with subtilisin BPN' or trypsin.The deletion of one residue (Gly66) caused increased inhibitoryactivity toward trypsin, indicating the protruding flexibleloop hinders binding with trypsin. Reduction of such hindranceby one-residue shortening in this loop is shown to be effectivefor the interaction of SSI(Lys73) with trypsin. In contrast,one-residue shortening had virtually no effect on inhibitiontoward subtilisin BPN'. Differences in the subsite structuresof these proteases may have been the reason for this contrast.The deletion of two residues (Thr64 and Gly66) in this regionconverted SSI into a temporary inhibitor. Structural analysisof the degradation intermediate showed that the peptide bondat the reactive site of doubly deleted SSI was cleaved by subtilisinBPN' after its binding with protease. Thus, the irreversibilityof the cleaved peptide bond at the reactive site of mutatedSSI in the complex with protease may possibly be the cause forits temporary inhibition. Irregular conformation around thereactive site caused by the deletion of two residues in theflexible loop would convert SSI into a temporary inhibitor.Thus, moderate flexibility in the flexible loop region may possiblybe a structural requirement for SSI to function.     

The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14{->}Lys and Asn14{->}Asp

A double-headed chymotrypsin inhibitor, WCI, from winged beanseeds was cloned for structural and biochemical studies. Theinhibitor was subjected to two point mutations at a conservedposition, Asn14. This residue, known to have a pivotal rolein stabilizing the first reactive-site loop (Gln63–Phe68)of the inhibitor, is highly conserved in the sequences of theother members of Kunitz (STI) family as well as in the sequencesof Kazal family of serine protease inhibitors. The mutants,N14K and N14D, were subjected to biochemical assay and theircharacteristics were compared with those of the recombinantinhibitor (rWCI). Crystallographic studies of the recombinantand the mutant proteins are discussed. These studies were primarilyaimed at understanding the importance of the protein scaffoldingtowards the conformational rigidity of the reactive-site loop.Our analysis reveals that, as the Lys14 side chain takes anunusual fold in N14K and the Asp14 side chain in N14D interactswith the loop residues by water-mediated hydrogen bonds, thecanonical conformation of the loop has remained effectivelyintact in both the mutant structures. However, minor alterationssuch as a 2-fold increase in the inhibitory affinity towardsthe cognate enzyme were observed.     

Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsininhibitor (Schistocerca gregaria chymotrypsin inhibitor) weredesigned and synthesized by convergent peptide synthesis. Foreach model peptide, the inhibitory constant (K_i) on chymotrypsinand the solution structure were determined. In addition, moleculardynamics calculations were performed for all of them. Two modelscontaining approximately half of the parent inhibitor (17 of35 residues) were designed and subsequently found to have nosubstantial inhibitory activity (K_i values in the mM range).The third model composed of 24 amino acid residues proved tobe an effective (K_i 10^–7) inhibitor of bovine chymotrypsin.Both the solution structure properties determined by NMR spectroscopyand the dynamic behaviour of the latter model system are comparableto the native inhibitor. In contrast, the structure and dynamicsof the first two related model peptides show characteristicdifferences. We suggest that the conformation and flexibilityof the modelled protease inhibitor are crucial for its biologicalefficiency. Moreover, the structural and dynamic features ofthe binding loop (28–33) and those of the rest of themolecule appear to be interdependent. Most importantly, thesestructural characteristics can be rationally modified, at leastpartially, by peptide design. Received March 7, 2003; revised August 25, 2003; accepted August 26, 2003.     

Decoding the Molecular Effects of Atovaquone Linked Resistant Mutations on Plasmodium falciparum Cytb-ISP Complex in the Phospholipid Bilayer Membrane

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins’ function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.     

Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulfur- to phosphate-containing substrates

Recent investigations have shown that the rhodanese domains,ubiquitous structural modules which might represent an exampleof conserved structures with possible functional diversity,are structurally related to the catalytic subunit of Cdc25 phosphataseenzymes. The major difference characterizing the active-siteof the Azotobacter vinelandii rhodanese RhdA, with respect tothe closely related Cdc25s (A, B, C), is that in Cdc25 phosphatasesthe active site loop [His–Cys–(X)₅–Arg] isone residue longer than in RhdA [His–Cys–(X)₄–Arg].According to the hypothesis that the length of the RhdA active-siteloop should play a key role in substrate recognition and catalyticactivity, RhdA scaffold was the starting point for producingmutants with single-residue insertion to generate the catalyticloop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analysesof the catalytic performances of the engineered RhdAs revealedthat elongation of the catalytic loop definitely compromisedthe ability to catalyze sulfur transfer reactions, while itgenerated ‘phosphatase’ enzymes able to interactproductively with the artificial substrate 3-O-methylfluoresceinphosphate. Although this study is restricted to an example ofrhodanese modules (RhdA), it provided experimental evidenceof the hypothesis that a specific mutational event (a single-residueinsertion or deletion in the active-site loop) could changethe selectivity from sulfur- to phosphate-containing substrates(or vice versa). Received February 17, 2003; revised May 30, 2003; accepted June 6, 2003.     

Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution

To expand the functionality of lipase B from Candida antarctica(CALB) we have used directed evolution to create CALB mutantswith improved resistance towards irreversible thermal inactivation.Two mutants, 23G5 and 195F1, were generated with over a 20-foldincrease in half-life at 70°C compared with the wild-typeCALB (WT-CALB). The increase in half-life was attributed toa lower propensity of the mutants to aggregate in the unfoldedstate and to an improved refolding. The first generation mutant,23G5, obtained by error-prone PCR, had two amino acid mutations,V210I and A281E. The second generation mutant, 195F1, derivedfrom 23G5 by error-prone PCR, had one additional mutation, V221D.Amino acid substitutions at positions 221 and 281 were determinedto be critical for lipase stability, while the residue at position210 had only a marginal effect. The catalytic efficiency ofthe mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyloctanoate was also found to be superior to that of WT-CALB. Received May 8, 2003; revised June 9, 2003; accepted June 23, 2003.     

Cold-adaptation mechanism of mutant enzymes of 3-isopropylmalate dehydrogenase from Thermus thermophilus

Random mutagenesis of Thermus thermophilus 3-isopropylmalatedehydrogenase revealed that a substitution of Val126Met in ahinge region caused a marked increase in specific activity,particularly at low temperatures, although the site is far fromthe binding residues for 3-isopropylmalate and NAD. To understandthe molecular mechanism, residue 126 was substituted with oneof eight other residues, Gly, Ala, Ser, Thr, Glu, Leu, Ile orPhe. Circular dichroism analyses revealed a decreased thermalstability of the mutants (T= 0–13°C), indicating structuralperturbations caused by steric conflict with surrounding residueshaving larger side chains. Kinetic parameters, k_cat and K_m valuesfor isopropylmalate and NAD, were also affected by the mutation,but the resulting k_cat/K_m values were similar to that of thewild-type enzyme, suggesting that the change in the catalyticproperty is caused by the change in free-energy level of theMichaelis complex state relative to that of the initial state.The kinetic parameters and activation enthalpy change (H) showedgood correlation with the van der Waals volume of residue 126.These results suggested that the artificial cold adaptation(enhancement of k_cat value at low temperatures) resulted fromthe destabilization of the ternary complex caused by the increasein the volume of the residue at position 126.     

Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

We have investigated factors affecting stability at the subunit–subunitinterface of the dimeric enzyme 3-isopropylmalate dehydrogenase(IPMDH) from Bacillus subtilis. Site-directed mutagenesis wasused to replace methionine 256, a key residue in the subunitinteraction, with other amino acids. Thermal stability againstirreversible inactivation of the mutated enzymes was examinedby analyzing the residual activity after heat treatment. Themutations M256V and M256A increased thermostability by 2.0 and6.0°C, respectively, whereas the mutations M256L and M256Ihad no effect. Thermostability of the M256F mutated enzyme was4.0°C lower than that of the wild-type enzyme. To our surprise,increasing the hydrophobicity of residue 256 within the hydrophobiccore of the enzyme resulted in a lower thermal stability. Themutated enzymes showed an inverse correlation between thermostabilityand the volume of the side chain at position 256. Based on theX-ray crystallographic structure of Escherichia coli IPMDH,the environment around M256 in the B.subtilis homolog is predictedto be sterically crowded. These results suggest that Met256prevents favorable packing. Introduction of a smaller aminoacid at position 256 improves the packing and stabilizes thedimeric structure of IPMDH. The van der Waals volume of theamino acid residue at the hydrophobic subunit interface is animportant factor for maintaining the stability of the subunit–subunitinterface and is not always optimized in the mesophilic IPMDHenzyme. Received September 3, 2002; revised June 13, 2003; accepted June 20, 2003.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

Glycine 85 of the trp-repressor of E.coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches

Experimental and computational analyses were performed on thecorepressor (L-tryptophan) binding site of the trp-repressorof Escherichia coli to investigate the ligandprotein interactions.Gly 85, one of the residues forming the hydrophobic pocket ofthe binding site, was systematically replaced with Ala, Val,Leu and Trp by cassette mutagenesis. Biochemical characterizationshowed that all these mutations caused significant decreasesin tryptophan binding activity. Free energy perturbation calculationswere performed for the mutants and were consistent with theexperimental results. The lack of a side chain at position 85was concluded to be essential for binding the corepressor; thestructure of the binding pocket was suggested to be tight inthe vicinity of Gly85.     

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study

A continuum electrostatics model is used to calculate the relativestabilities of 117 mutants of staphylococcal nuclease (SNase)involving the mutation of a charged residue to an unchargedresidue. The calculations are based on the crystallographicstructure of the wild-type protein and attempt to take implicitlyinto account the effect of the mutations in the denatured stateby assuming a linear relationship between the free energy changescaused by the mutation in the native and denatured states. Agood correlation (linear correlation coefficient of 0.8) isfound with published experimental relative stabilities of thesemutants. The results suggest that in the case of SNase (i) chargedresidues contribute to the stability of the native state mainlythrough electrostatic interactions, and (ii) native-like electrostaticinteractions may persist in the denatured state. The continuumelectrostatics method is only moderately sensitive to modelparameters and leads to quasi-predictive results for the relativemutant stabilities (error of 2–3 kJ mol^–1 or ofthe order of k_BT), except for mutants in which a charged residueis mutated to glycine. Received March 24, 2003; revised August 11, 2003; accepted September 12, 2003.     

Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyrand B27Thr, buried in the dimer of insulin, were identifiedby means of alanine-scanning mutagenesis. The receptor bindingactivity, in vivo biological potency and self-association propertiesof the seven single alanine human insulin mutants were determined.Four of the seven single alanine mutants, [B12Ala]human insulin,[B16Ala]human insulin, [B24Ala]human insulin and [B26Ala]humaninsulin, are monomeric insulin, which indicates that B12Val,B16Tyr, B24Phe and B26Tyr are crucial for the formation of insulindimer. The monomeric [B16Ala]human insulin and [B26Ala]humaninsulin retain 27 and 54% receptor binding activity, respectively,and nearly the same in vivo biological potency compared withnative insulin, so they could be developed as the fast-actinginsulin.     

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.      

A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization

A mechanism by which ligand binding to the extracellular domainof a growth factor receptor causes activation of its cytoplasmictyrosine kinase domain is that binding promotes receptor dimerization.Recently we proposed a model in which dimerization of the transmembrane-helices in one member of this family, rat neu, is mediatedby the presence of three specific residues. This paper showsthat a similar sequence motif is observed in 18 of the 20 transmembrane-helices of the tyrosine kinase family of growth factor receptors.The motif encompasses a five residue segment in which position0 (P0) requires a small side chain (Gly, Ala, Ser, Thr or Pro),P3 an aliphatic side chain (Ala, Val, Leu or Ile) and P4 onlythe smallest side chains (Gly or Ala). In addition other featuresof the transmembrane sequences are reported. It is concludedthat the dimerization of transmembrane -helices may be a generalmechanism of tyrosine kinase activation in this family of growthfactor receptors.     

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase

A mutant of Lactobacillus casei dihydrofolate reductase hasbeen constructed in which Thr63, a residue which interacts withthe 2'-phosphate group of the bound coenzyme, is replaced byalanine. This substitution does not affect k_cat, but producesan 800-fold increase in the K_m for NADPH, which reflects dissociationof NADPH from the enzyme-NADPH-tetrahydrofolate complex, anda 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociationconstant for the enzyme-NADPH complex. The difference in magnitudeof these effects indicates a small effect of the substitutionon the negative cooperativity between NADPH and tetrahydrofolate.Stopped-flow studies of the kinetics of NADPH binding show thatthe weaker binding arises predominantly from a decrease in theassociation rate constant. NMR spectroscopy was used to comparethe structures of the mutant and wild-type enzymes in solution,in their complexes with methotrexate and with methotrexate andNADPH. This showed that only minimal structural changes resultfrom the mutation; a total of 47 residues were monitored fromtheir resolved ¹H resonances, and of these nine in the binarycomplex and six in the ternary differed in chemical shift betweenmutant and wild-type enzyme. These affected residues are confinedto the immediate vicinity of residue 63. There is a substantialdifference in the ³¹P chemical shift of the 2'-phosphate ofthe bound coenzyme, reflecting the loss of the interaction withthe side chain of Thr63. The only changes in nuclear Overhausereffects (NOEs) observed were decreases in the intensity of NOEsbetween protons of the adenine ring of the bound coenzyme andthe nearby residues Leu62 and Ile102, showing that the substitutionof Thr63 does cause a change in the position or orientationof the adenine ring in its binding site.     

Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies

To investigate the contribution of tryptophan-121 (Trp121) residueto the structure and function of soluble Cu_A domain of cytochromec oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deletedmutant of the soluble domain of Paracoccus versutus cytochromec oxidase, were constructed and expressed in Escherichia coliBL21 (DE3). Optical spectral studies showed that both the coordinationstructure of the Cu_A center and the secondary structure of theprotein were changed significantly in the Leu substitution anddeletion mutants of Trp121. Their electron transfer activitywith cytochrome c was inhibited severely, as shown in stopped-flowkinetic studies. However, the Cu_A center can be reconstructedin the Trp121Tyr mutant although its stability decreases comparedwith the wild-type protein. This mutant keeps the same secondarystructure as the wild-type protein, but can only transfer electronswith cytochrome c at a rate of one-seventh-fold. Based on theinformation on the structure, we also investigated and analyzedthe possible factors that affect electron transfer. It appearsthat the aromatic ring, the size of the side chain and the hydrogenbonding ability of the Trp121 are crucial to the structure andfunction of the soluble Cu_A domain. Received September 11, 2002; revised February 26, 2003; accepted May 27, 2003.     

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A₂ (PLA₂) islocated at the entrance to the active site. To study the roleof residue 31 in PLA₂, six mutant enzymes were produced by site-directedmutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Seror Gly. Direct binding studies indicated a three to six timesgreater affinity of the Trp31 PLA₂ for both monomeric and micellarsubstrate analogs, relative to the wild-type enzyme. The otherfive mutants possess an unchanged affinity for monomers of theproduct analog n-decylphosphocholine and for micelles of thediacyl substrate analog rac-l,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine.The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholinewere decreased 9–20 times for these five mutants. Kineticstudies with monomeric substrates showed that the mutants haveV_max values which range between 15 and 70% relative to the wild-typeenzyme. The V_max values for micelles of the zwitterionic substratel,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3–50times. The K_m values for the monomeric substrate and the k_mvalues for the micellar substrate were hardly affected in thecase of five of the six mutants, but were considerably decreasedwhen Trp was present at position 31. The results of these investigationspoint to a versatile role for the residue at position 31: involvementin the binding and orientating of monomeric substrate (analogs),involvement in the binding of the enzyme to micellar substrateanalogs and possibly involvement in shielding the active sitefrom excess water.     

Thermosensitive mutants of Aspergillus awamori glucoamylase by random mutagenesis: inactivation kinetics and structural interpretation

Abstract Seven thermosensitive glucoamylase mutants generated by randommutagenesis and expressed inSaccharomyces cerevisiae were sequencedand their inactivation kinetics were determined. Wild-type glucoamylaseexpressed in S.cerevisiae was more glycosylated and more stablethan the native Aspergillus niger enzyme. All mutants had lowerfree energies of inactivation than wild-type glucoamylase. Inthe Ala39 Val, Ala302 Val and Leu410 Phe mutants, small hydrophobicresidues were replaced by larger ones, showing that increasesin size and hydrophobicity of residues included in hydrophobicclusters were destabilizing. The Gly396 Ser and Gly407 Aspmutants had very flexible residues replaced by more rigid ones,and this probably induced changes in the backbone conformationthat destabilized the protein. The Prol28 Ser mutation changeda rigid residue in an a-helix to a more flexible one, and destabilizedthe protein by increasing the entropy of the unfolded state.The Ala residue in the Ala442 Thr mutation is in the highlyO-glycosylated region surrounded by hydrophilk residues, whereitmay be a hydrophobic anchor Unking the O-glycosylated arm tothe catalytic core. It was replaced by a residue that potentiallyis O-glycosylated. In five of the seven mutations, residuesthat were part of hydrophobic microdomains were changed, confirmingthe importance of the latter in protein stability and structure     

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome P450 monooxygenase

Furanocoumarins represent plant toxins that are used in thetreatment of a variety of skin diseases and are metabolizedby cytochrome P450 monooxygenases (P450s) existing in insectssuch as Papilio polyxenes (the black swallowtail). To elucidatethe active site in the CYP6B1 protein that is the principalP450 existing in this species, we have constructed a homologymodel of it based on sequence and structure alignments withthe bacterial CYP102 protein whose crystal structure has beendefined and with the insect CYP6B4 protein that also metabolizesfuranocoumarins. In the derived CYP6B1 model, Phe116 and His117in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to theformation of a resonant network that stabilizes the P450’scatalytic site and allows for interactions with its furanocoumarinsubstrates. The first two of these residues are absolutely conservedin all members of the insect CYP6B subfamily and the last twoare variable in different members of the CYP6B subfamily. Acombination of theoretical and experimental docking analysesof two substrates (xanthotoxin and bergapten) and two inhibitors(coumarin and pilocarpine) of this P450 provide significantinformation on the positioning of furanocoumarins within thiscatalytic pocket. Molecular replacement models based on theresults of variations at two of these critical amino acids providesupport for our furanocoumarin-docked model and begin to rationalizethe altered substrate reactivities observed in experimentalanalyses. Received December 4, 2002; revised June 8, 2003; accepted June 23, 2003.