Protein engineering of alcohol dehydrogenases; effects of amino acid changes at positions 93 and 48 of yeast ADH1

By protein engineering we have investigated changes to two aminoacid residues (Trp93 and Ser48) in the substrate pocket of yeastalcohol dehydrogenase 1. Upon changing Thr48 to serine we producedan enzyme which has markedly greater activity towards aliphaticalcohols with chain length up to 8, together with a generalincrease in catalytic activity (V/K). Changes at position 93were less pronounced, with the Phe enzyme being more activethan the parent towards the range of alcohols but with the alanineenzyme showing very little difference from the wild-type. Enzymeswith the double changes at 48 and 93 showed increased activitytowards alcohols with 3–8 carbons but the increases werenot additive over the single changes. The enzymes with changesat the two positions would metabolize both stereoisomers of2-octanol whereas the parent ADH would attack only one of them.None of the engineered enzymes would attack cyclohexanol oraromatic alcohols. The results are in general agreement withthe prediction that reducing the size of amino acids in thesubstrate pocket would enhance the ability to oxidize alcoholslarger than ethanol.     

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.     

Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45

Using point mutated overproducing strains of E.coli, ribonucleaseT1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp,Tyr45Trp or Trp59Tyr and the corresponding double substitutionsTyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr.Steady state kinetics of the transesterification reaction forthe two dinucleoside monophosphate substrates guanylyl-3', 5'-cytidineand guanylyl-3', 5'-adenosine indicate that the tryptophan canbe introduced in different positions within the ribonucleaseT1 molecule without abolishing enzymatic activity. The Trp59Tyrexchange even enhances catalysis of the cleavage reaction (k_cat/K_m)relative to the wild type enzyme and similar effects are foundwith single tyrosine to tryptophan substitutions. For the pHdependencies of the guanylyl-3', 5'-cytidine transesterificationreaction of wild type ribonuclease T1 and of the variants, typicallybell-shaped curves are observed with a plateau in the rangepH 4.5–7.0. Their shapes and slopes indicate that theenzymes are comparable in their macroscopic pK_a, values. AtpH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-foldhigher transesterification activity for guanylyl-3', 5'-adenosineand a 2-fold increase for guanylyl-3', 5'-cytidine comparedto the wild type enzyme, i.e. this variant catalyses the transesterificationof the substrate guanylyl-3', 5'-adenosine with the same orbetter efficiency as guanylyl-3', 5'-cytidine.     

Lysozyme requires fluctuation of the active site for the manifestation of activity

Mutations around His15 which lie far away from the active site,stimulated glycol chitin activity of lysozyme at physiologicaltemperature. Del-Argl4Hisl5 lysozyme, a mutant lysozyme whoseArgl4 and Hisl5 were deleted together, and has the highest activityamong these mutant lysozymes, had a similar binding abilityto a trimer of N-acetyl-glucosamine, a substrate analogue, relativeto native lysozyme. This suggests that the increased activitywas due to an increased k_cat in the catalysis reaction. TheH-D exchange rate of the N-1 proton in the Trp63 which is locatedin the active site cleft, was enhanced in the Del-Argl4Hisl5lysozyme, while 2-D proton NMR analysis revealed no conformationalchange around Trp63. We conclude that some sort of fluctuationat the active site might be required for the manifestation ofactivity. This theory is supported by the finding that the Del-Argl4Hisl5lysozyme showed a shift in temperature dependency of activityto lower temperatures compared with that of native lysozyme.     

Fluorescent properties of the Escherichia coli D-xylose isomerase active site

The consequences of active site mutations of the Escherichiacoli D-xylose isomerase (E.C. 5.3.1.5 [EC] ) on substrate bindingwere examined by fluorescence spectroscopy. Site-directed mutagenesisof conserved tryptophan residues in the E.coli enzyme (Trp49and Trpl88) reveals that fluorescence quenching of these residuesoccurs during the binding of xylose by the wild-type enzyme.The fluorescent properties of additional active site substitutionsat His101 were also examined. Substitutions of His101 whichinactivate the enzyme were shown to have altered spectral characteristics,which preclude detection of substrate binding. In the case ofH101S, a mutant protein with measurable isomerizing activity,substrate binding with novel fluorescent properties was observed,possibly the bound pyranose form of xylose under steady-stateconditions.     

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase

Platelet-activating factor acetylhydrolases (PAF-AHs) are uniquePLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipidswith a marked preference for very short acyl chains, typicallyacetyl. The recent solution of the crystal structure of the₁ catalytic subunit of isoform Ib of bovine brain intracellularPAF-AH at 1.7 Å resolution paved the way for a detailedexamination of the molecular basis of substrate specificityin this enzyme. The crystal structure suggests that the sidechains of Thr103, Leu48 and Leu194 are involved in substraterecognition. Three single site mutants (L48A, T103S and L194A)were overexpressed and their structures were solved to 2.3 Åresolution or better by X-ray diffraction methods. Enzyme kineticsshowed that, compared with wild-type protein, all three mutantshave higher relative activity against phospholipids with sn-2acyl chains longer than an acetyl. However, for each of themutants we observed an unexpected and substantial reductionin the V_max of the reaction. These results are consistent withthe model in which residues Leu48, Thr103 and Leu194 indeedcontribute to substrate specificity and in addition suggestthat the integrity of the specificity pocket is critical forthe expression of full catalytic function, thus conferring veryhigh substrate selectivity on the enzyme.     

FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolylisomerase that binds the macrolides FK506 and rapamycin. Wehave examined the role of the binding pocket residues of FKBP12in protein–ligand interactions by making conservativesubstitutions of 12 of these residues by site-directed mutagenesis.For each mutant FKBP12, we measured the affinity for FK506 andrapamycin and the catalytic efficiency in the cis–transpeptidyl-prolyl isomerase reaction. The mutation of Trp59 orPhe99 generates an FKBP12 with a significantly lower affinityfor FK506 than wild-type protein. Tyr26 and Tyr82 mutants areenzymatically active, demonstrating that hydrogen bonding bythese residues is not required for catalysis of the cis–transpeptidyl-prolyl isomerase reaction, although these mutationsalter the substrate specificity of the enzyme. We conclude thathydrophobic interactions in the active site dominate in thestabilization of FKBP12 binding to macrolide ligands and tothe twisted-amide peptidyl-prolyl substrate intermediate.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis

Site-specific mutagenesis was employed to study structure-functionrelationships at the substrate binding site of rat tissue kallikrein.Four kallikrein mutants, the Pro219 deletion (P219del), the34–38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34–38)IN],the Trp215Gly exchange (W215G) and the double mutant with Tyr99Hisand Trp215Gly exchange (Y99H:W215G) were created by site-directedmutagenesis to probe their function in substrate binding. Themutant proteins were expressed in Esclzerichia coli at highlevels and analyzed by Western blot. These mutant enzymes werepurified to apparent homogeneity. Each migrated as a singleband on SDS-PAGE, with slightly lower molecular mass (36 kDa)than that of the native enzyme, (38 kDa) because of their lackof glycosylation. The recombinant kallikreins are immunologicallyidentical to the native enzyme, displaying parallelism withthe native enzyme in a direct radioimmunoassay for rat tissuekallikrein. Kinetic analyses of K_m and k_cat using fluorogenicpeptide substrates support the hypothesis that the Tyr99–Trp215interaction is a major determinant for hydrophobic P2 specificity.The results suggest an important role for the 34–38 loopin hydrophobic P3 affinity and further show that Pro219 is essentialto substrate binding and efficient catalysis of tissue kallikrein.     

The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosalipase for the hydrolytic activity at the water/lipid interfacewas investigated by site-directed mutagenesis. It was foundthat the effect on the hydrolytic activity upon the replacementof Trp89 with Phe, Leu, Gly or Glu was substrate dependent TheTrp89 mutants displayed an altered chain length specificitytowards triglycerides, with a higher relative activity towardstriacetin and trioctanoin compared with tributyrin. Trp89 wasshown to be lessimportant in the hydrolysis of vinyl esterscompared with ethylesters and triglycerides. An exclusive effecton the acylation reaction rate by the mutation of Trp89 wasconsistent with the data. It is suggested that Trp89 is importantin the process of binding the acyl chain of thesubstrate intothe activesite for optimal acylation reaction rate. The Trp89Phemutation resulted in an increased hydrolytic activity towards2-alkylalkanoic acid esters. This is suggested to be due toreduction of unfavourable van der Waals contacts between Trp89and the 2-substituent of the substrate. Thus, in contrast tonatural substrates, Trp89 has a negative impact on the catalyticefficiencywhen substrates with bulky acyl chains are used. Incontrast to the Trp89 mutations, the effect on the hydrolyticactivity of the Glu87Ala mutation was almost substrate independent,35–70% activity of wild-type lipase. Areduction of boththe acylation and deacylation reaction was consistent with thedata.     

EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodexynucleotides

Synthetic oligodeoxynucleotides with single methyl phosphonate(mp) substitutions were used for an analysis of the contributionof phosphate contacts to the recognition of the cleavage siteby the restriction endonuclease EcoRV. Only in the last positionwithin the recognition sequence, is the methyl phosphonate substitutiontolerated by the enzyme. The wild-type enzyme cleaves the S_Pdiastereomer of the oligodeoxynucleotide GACGATAT_mpCGTC andthe unmodified sequence with equal rates, whereas the R_P diastereomeris cleaved much more slowly. Inspection of the crystal structureof an EcoRV–DNA complex revealed that the non-bridgingoxygen atoms of the phosphodiester bond between the T and Cbases are in hydrogen bonding distance of the hydroxyl groupof the amino acid Thr94. We therefore tried to engineer a variantof EcoRV that would prefer a methyl phosphonate linkage overa normal phosphodiester bond and produced mutants with aminoacid exchanges at position 94. One of them, Thr94Val, showsa dramatically reduced activity towards the unmodified DNA anddoes not accept the R_p diastereomer, but cleaves the S_P diastereomerwith the same rate as wild-type EcoRV. Its selectivity, i.e.the ratio of cleavage rates determined for the unmodified andmodified substrates, differs by three orders of magnitude fromthat of the wild-type enzyme.     

Replacing the glutamate ligand in the structural zinc site of Sulfolobus solfataricus alcohol dehydrogenase with a cysteine decreases thermostability

The alcohol dehydrogenase gene from the thermophilic archaeumSulfolobus solfataricus has been subcloned and expressed inEscherichia coli under the control of the T7 inducible promoter.Therecombinant protein shows properties analogous to those of thenative enzyme, including thermostability, despite the fact thatE.coli does not post-translationally modify two lysine residueswhich are N--methylated in the native enzyme. We constructeda 3-D model of the S.solfataricus alcohol dehydrogenase usingthe known structure of its isozyme from horse liver as a template.Our analysis of the structural zinc binding site suggested thatthis site is present andfunctional in the S.solfataricus enzymeand that a glutamate ligand can contribute to thermostabilityby influencing electrostatic interactions around the metal centre.To investigate thishypothesis, we constructed, expressed andcharacterized a mutant where the glutamate is replaced by acysteine, thus restoring the zinc binding site of mesophilicalcohol dehydrogenases. Themutant shows the same activity buta reduced thermostability with respect to the wild-type recombinantprotein, as suggested by our model.     

Mutations in the FAD-binding fold of alcohol oxidase from Hansenula polymorpha

Alcohol oxidase of methylotrophic yeast is an FAD-containingenzyme. When in its active form, the enzyme is an octamer andlocated in the peroxisomes. To study the importance of FAD-bindingon the activity, octamerization and intracellular localizationof the enzyme, alcohol oxidase of Hansenula polymorpha was mutatedin its presumed nucleotide-binding domain, which is formed bythe N-terminal sequence. Whereas mutations of a glutamic acidresidue (E42) reduced thestability of the octamer, it hardlyaffected enzyme activity and expression. However, replacementsof three conserved glycines (G13, G15 and G18) and a conservedglutamic acid (E39) within the fold had severe effects. The utations not only resulted in loss of enzyme activity but inreduced protein levels as well, probably due to decreased stabilityof the mutant alcohol oxidase. However, octamerization of theprotein still occurred. The existence of inactive octamericproteins provides information about the formation pathway ofthis octameric flavoprotein.     

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M·EcoRV) is an -adeninemethyltransferase. We have used two different programs to predictthe secondary structure of M·EcoRV. The resulting consensusprediction was tested by a mutant profiling analysis. 29 neutralmutations of M·EcoRV were generated by five cycles ofrandom mutagenesis and selection for active variants to increasethe reliability of the prediction and to get a secondary structureprediction for some ambiguously predicted regions. The predictedconsensus secondary structure elements could be aligned to thecommon topology of the structures of the catalytic domains ofM·HhaI and M·TaqI. In a complementary approachwe have isolated nine catalytically inactive single mutants.Five of these mutants contain an amino acid exchange withinthe catalytic domain of M·EcoRV (Val20-Ala, Lys81Arg,Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant bindsDNA similarly to wild-type M·EcoRV, but is catalyticallyinactive. Hence this mutant behaves like a bona fide activesite mutant. According to the structure prediction, Trp231 islocated in a loop at the putative active site of M·EcoRV.The other inactive mutants were insoluble. They contain aminoacid exchanges within the conserved amino acid motifs X, IIIor IV in M·EcoRV confirming the importance of these regions.     

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.     

Activity of linked HIV-1 proteinase dimers containing mutations in the active site region

Mutations were introduced into the active site triplet (Asp–Thr–Gly)of one or both subunits of a linked dimer of human immunodeficiencyvirus type 1 proteinase. Mutation of Thr to Ser in one or bothsubunits did not alter the activity of the enzyme substantially,whereas its mutation to Asn in one subunit caused a dramaticdecrease in catalytic efficiency. Mutation of Gly to Val inone subunit also yielded an enzyme with very low activity. Theenzymes containing Thr Asn and Gly Val mutations in both subunitsresulted in inactive enzymes, based on their inability to self-processand on assay with an oligopeptide substrate. The dramatic decreasein enzyme efficiency of the mutants was interpreted using molecularmodels of the enzymes.     

Probing the role of threonine and serine residues of E.coli asparaginase II by site-specific mutagenesis

Site-specific mutagenesis has been used to probe amino acidresidues proposed to be critical in catalysis by Escherichiacoli asparaginase II. Thr12 is conserved in all known asparaginases.The catalytic constant of a T12A mutant towards L-aspartk acidß-hydroxamate was reduced to 0.04% of wild type activity,while its An, and stability against urea denaturation were unchanged.The mutant enzyme T12S exhibited almost normal activity butaltered substrate specificity. Replacement of Thr119 with Alaled to a 90% decrease of activity without markedly affectingsubstrate binding. The mutant enzyme S122A showed normal catalyticfunction but impaired stability in urea solutions. These dataindicate that the hydroxyl group of Thr12 is directly involvedin catalysis, probably by favorably interacting with a transitionstate or intermediate. By contrast, Thr119 and Ser122, bothputative target sites of the inactivator DONV, are functionallyless important.     

Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease

On the basis of the homology with the Bacillus thermoproteolyticuszinc endopeptidase thermotysin, we hypothesized that Glu-143and His-231 are the key residues for the catalytic activityof the Bacillus subtilis neutral protease. To test this possibilityby site-directed mutagenesis, we substituted these two residueswith Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively.All these substitutions dramatically affected the amount ofsecreted mutant proteins, as determined by immunological methods,and their catalytic activities. No appreciable secretion wasobserved with the three Glu mutants Trp, Ser and Arg, whereasthe Glu–Ala mutant enzyme was secreted at a level of afew hundred micrograms per litre of culture. The His mutantswere all secreted at higher levels (in the order of a few milligramsper litre) and their residual catalytic activity could be determinedusing Z-Ala-Leu-Ala as substrate. Our results confirm the keyrole played by Glu-143 and His-231 in catalysis and moreoversuggest the existence of a relationship between the catalyticactivity of the enzyme and the extent of its secretion. In thiscontext, we present data suggesting an autoproteolytic mechanismof cleavage of the precursor form of the enzyme, analogous tothe one previously reported for the B.subtilis subtilisin.     

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.     

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor

The protein kinase family can be subdivided into two main groupsbased on their ability to phosphorylate Ser/Thr or Tyr substrates.In order to understand the basis of this functional difference,we have carried out a comparative analysis of sequence conservationwithin and between the Ser/Thr and Tyr protein kinases. A multiplesequence alignment of 86 protein kinase sequences was generated.For each position in the alignment we have computed the conservationof residue type in the Ser/Thr, in the Tyr and in both of thekinase subfamilies. To understand the structural and/or functionalbasis for the conservation, we have mapped these conservationproperties onto the backbone of the recently determined structureof the cAMP–dependent Ser/Thr kinase. The results showthat the kinase structure can be roughly segregated, based uponconservation, into three zones. The inner zone contains residueshighly conserved in all the kinase family and describes thehydrophobic core of the enzyme together with residues essentialfor substrate and ATP binding and catalysis. The outer zonecontains residues highly variable in all kinases and representsthe solvent–exposed surface of the protein. The thirdzone is comprised of residues conserved in either the Ser/Thror Tyr kinases or in both, but which are not conserved betweenthem. These are sandwiched between the hydrophobic core andthe solvent-exposed surface. In addition to analyzing overallconservation hi the kinase family, we have also looked at conservationof its substrate and ATP binding sites. The ATP site is highlyconserved throughout the kinases, whereas the substrate bindingsite is more variable. The active site contains several positionswhich differ between the Ser/Thr and Tyr kinases and may beresponsible for discriminating between hydroxyl bearing sidechains. Using this information we propose a model for Tyr substratebinding to the catalytic domain of the epidermal growth factorreceptor (EGFR).