Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniqueswere generated to analyze the structure–function relationshipin this characteristic aspartk proteinase. In order to preparethe mutant enzymes in their active form, we established proceduresfor purification of correctly refolded prochymosin from inclusionbodies produced in Escherichia coli transformants and for itssubsequent activation. Mutagenesis by linker insertion intocDNA produced several mutants with an altered ratio of milkclotting activity to proteolytic activity and a different extentof stability. In addition to these mutants, several mutantswith a single amino acid exchange were also constructed by site-directedmutagenesis and kinetic parameters of these mutant enzymes weredetermined by using synthetic hexa- and octa-peptides as substrates.Exchange of Tyr75 on the flap of the enzyme to Phe caused amarked change of substrate specificity due to the change ofk_cat or K_m, depending on the substrate used. Exchange of Val110and Phe111 also caused a change of kinetic parameters, whichindicates functional involvement of these hydrophobic residuesin both the catalytic function and substrate binding. The mutantLys220–Leu showed a marked shift of the optimum pH tothe acidic side for hydrolysis of acid-denatured haemoglobinalong with a distinct increase in k_cat for the octa-peptidein a wide pH range.     

Protein engineering loops in aspartic proteinases: site-directed mutagenesis, biochemical characterization and X-ray analysis of chymosin with a replaced loop from rhizopuspepsin

The loop exchange mutant chymosm 155–164 rhizopuspepsinwas expressed in Trichoderma reesei and exported into the mediumto yield a correctly folded and active product. The biochemicalcharacterization and crystal structure determination at 2.5Å resolution confirm that the mutant enzyme adopts a nativefold. However, the conformation of the mutated loop is unlikethat in native rhizopuspepsin and involves the chelation ofa water molecule in the loop. Kinetic analysis using two syntheticpeptide substrates (six and 15 residues long) and the naturalsubstrate, milk, revealed a reduction in the activity of themutant enzyme with respect to the native when acting on boththe long peptide substrate and milk. This may be a consequenceof the different charge distribution of the mutated loop, itsincreased size and/or its different conformation.     

Site-directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin

The functional contributions of amino acid residues Thr218 andAsp304 of chymosin, both of which are highly conserved in theaspartic proteinases, are analysed by means of site-directedmutagenesis. The optimum pH values, milk-clotting (C) and proteolytic(P) activities and kinetic parameters for synthetic oligopeptidesas substrates were examined for the mutant enzymes. The mutationThr2l8Ser caused a marked increase in the C/P ratio, which seemedto be due to a change in substrate recognition. Although thenegative charge of Asp304 had been expected to play a role inlowering the optimum pH values in the aspartic proteinases,this turned out not to be the case in chymosin because boththe mutations Asp304Ala and Asp304Glu caused a similar shiftof the optimum pH towards the acidic side. In addition, themutation Lys220Leu, which we generated previously, was foundto cause a decrease in the C/P ratio, mainly due to the increasein the proteolytic activity.     

Redesign of the substrate specificity of human cathepsin D: the dominant role of position 287 in the S2 subsite

Interest in the active site specificity of human cathepsin Dstems from the search for specific therapeutic agents againstmany of the sequentially and structurally homologous membersofthe aspartic proteinase family. The work presented here examinedone amino acid in the cathepsin D sequence, located in the S₂subsite, which contributes substantially to the specificityof enzyme-Ugand interactions at the enzyme active site. Previousstudies reported on the specificity of binding and catalysisby native and recombinant human cathepsin D explored throughkinetic studies using a systematic series of synthetic substrates.Utilizing a rulebased molecular model of human cathepsin D,Met287 was suggested as a candidate for mutagenesis to furtherexplore selectivity within the S₂ subsite of the cathepsin Dactive site. Met287 mutant derivatives of human cathepsin Dwere designed, expressed and characterized in kineticstudies.Native cathepsin D accommodates large hydrophobic residues inthe P₂ position of a substrate; positively charged residuesin P₂ are not favorable for catalysis.It was demonstrated thataltering Met287 of human cathepsin D to more polar amino acidsproduced active mutant enzymes with significantly altered substratespecificity.     

Involvement of a residue at position 75 in the catalytic mechanism of a fungal aspartic proteinase, Rhizomucor pusilus pepsin. Replacement of tyrosine 75 on the flap by asparagine enhances catalytic efficiency

Residue 75 on the flap, a beta hairpin loop that partially coversthe active site cleft, is tyrosine in most members of the asparticproteinase family. Site-directed mutagenesis was carried outto investigate the functional role of this residue in Rhizomucorpusilus pepsin, an aspartic proteinase with high milk-clottingactivity produced by the fungus Rhizomucor pusillus. A set ofmutated enzymes with replacement of the amino acid at position75 by 17 other amino acid residues except for His and Gly wasconstructed and their enzymatic properties were examined. Strongactivity, higher than that of the wild-type enzyme, was foundin the mutant with asparagine (Tyr75Asn), while weak but distinctactivity was observed in Tyr75Phe. All the other mutants showedmarkedly decreased or negligible activity, less than 1/1000of that of the wild-type enzyme. Kinetic analysis of Tyr75Asnusing a chromogenic synthetic oligopeptide as a substrate revealeda marked increase in k_cat with slight change in K_m, resultingin a 5.6-fold increase in k_cat/k_m. When differential absorptionspectra upon addition of pepstatin, a specific inhibitor foraspartic proteinase, were compared between the wild-type andmutant enzymes, the wild-type enzyme and Tyr75Asn, showing strongactivity, had spectra with absorption maxima at 280, 287 and293 nm, whereas the others, showing decreased or negligibleactivity, had spectra with only two maxima at 282 and 288 nm.This suggests a different mode of the inhibitor binding in thelatter mutants. These observations suggest a crucial role ofthe residue at position 75 in enhancing the catalytic efficiencythrough affecting the mode of substrate-binding in the asparticproteinases.     

Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'₁, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (k_{cat/Km)FA-A-R-OH/(kcatm})_FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below.     

Crystallographic phases through genetic engineering: experiences with colicin A

Colicins are antibiotic proteins that kill sensitive Escherichiacoli cells. The structure of the pore-forming fragment of colicinA has been solved to 2.5 A resolution using the techniques ofX-ray crystallography and genetic engineering. Site-directedmutagenesis was used to construct a number of cysteine-containingmutant proteins, one of which yielded an excellent mercurialderivative. Our experiences suggest strategies for obtaininguseful heavy-atom derivatives for protein crystallography usinggenetic engineering techniques.     

Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1

The evolutionally conserved aspartyl residues (Asp57, Asp98and Asp152) in human glutathione S-transferase P1-1 were replacedwith alanine by site-directed mutagenesis to obtain the mutants(D57A, D98A and D152A). The replacement of Asp98 with alanineresulted in a decrease of the affinity for S-hexyl-GSH-agarose,a 5.5-fold increase of the K_m^GHS and a 2.9-fold increase ofthe I₅₀ of S-hexyl-GSH for GSH–CDNB conjugation. Asp98seems to participate in the binding of GSH through hydrogenbonding with the -carboxylate of the -glutamyl residue of GSH.The k_cat of D98A was 2.6-fold smaller than that of the wild-type,and the pK_a of the thiol group of GSH bound in D98A was {smalltilde}0.8 pK units higher than those in the wild-type. Asp98also seems to contribute to the activation of GSH to some extent.On the other hand, most of the kinetic parameters of D57A andD152A were similar to those of the wild-type. However, the thermostabilitiesof D57A and D152A were significantly lower than that of thewild-type. Asp57 and Asp152 seem to be important for maintainingthe proper conformation of the enzyme.     

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK,has been constructed which greatly facilitates the insertionand removal of mutant enzyme genes by cleavage at newly introducedBamtHI sites. This vector has been used to prepare mutant proteinin appreciable (100 mg) quantities for use in kinetic, crystaUographicand NMR experiments. Aspartate 372 is an invariant amino acidresidue in genes known to code for a functionally active PGK.The function of this acidic residue appears to be to help desolvatethe magnesium ion compfexed with either ADP or ATP when thissubstrate binds to the enzyme. Both crystallographk and nuclearmagnetic resonance experiments show that the replacement ofthe residue with asparagine has only minimal effects on theoverall structure. The substitution of the charged carboxylgroup with that of the neutral amide affects the binding ofthe nucleotide substrate as predicted but not, as might havebeen expected, the binding of 3-phospho-glycerate. The overallvelocity of the enzymic reaction (V_max) is reduced 10-fold bythe substitution of aspartic acid 372 by an asparagine residue(D372N). This reduction in V_max is considerably less than onewould expect from its known position within the structure ofthe enzyme. This result therefore poses questions about ourunderstanding of charged groups at the active centres of enzymesand of the reason for their apparent conservation.     

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.     

Chemical modification and site-directed mutagenesis of Tyr36 of 3-isopropylmalate dehydrogenase from Thermits thermophUus HB8

3-Isopropylmalate dehydrogenase from an extreme thermo-phile,Thermus thermophUus HB8, was chemically modified with tetranitromethanewhich nitrated 1.5-2.0 Tyr residues per subunit. The nitrationwas biphask and parallel to the loss of activity. The modifiedresidue in the first phase was identified to be Tyr36, whichis distantly located from the active site of the enzyme. Thefunction of Tyr36 was investigated by site-specific replacementwith Phe. The Michaelis constant for the substrate or co-enzymewas not altered by the replacement, whereas the catalytic constantdecreased down to -5%. X-ray analysis of the mutant enzyme revealedthat Arg94 moved the largest distance among the active siteresidues, that is, the NH1 and NH2 of the guanidino group moved1.11 and 1.32 Å respectively. The results suggest thatArg94 is responsible for the enzyme catalysis     

Identification by site-directed mutagenesis of amino acids in the subsite of bovine pancreatic ribonuclease A

In addition to hydrolysing RNA, bovine pancreatic ribonucleasesplits esters of pyrimidine nucleoside 3'-phosphates, includingdinucleotides. For a series of 3':5'-linked dinucleotides ofgeneral structure CpN, where N is a 5' linked nucleoside, k_calfor the release of N varies enormously with the precise structureof N. Structural studies have been interpreted to indicate thatthe group N interacts with a subsite, B2, on the enzyme thatcomprises Gln69, Asn71 and Glulll. We report studies by site-directedmutagenesis that indicate that Gln69 is not involved in productiveinteractions with any of the dinucleotide substrates and thatAsn71 is an important component of subsite B2 for all dinucleotidesubstrates tested. Glulll appears to be functionally involvedin catalysis for dinucleotide substrates solely when N is guanosine.     

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis inan attempt to increase the cytotoxic potency and receptor-bindingaffinity of the diphtheria-toxin-related interleukin-2 (IL-2)fusion toxins. Previous studies have demonstrated that boththe DAB₄₈₆-IL-Z and DAB₃₈₉-IL-2 forms of the fusion toxin consistof three functional domains: the N-tenninal fragment-A-assodatedADP-ribosyltransferase, the hydrophobk-membrane-associatingdomains, and the C-terminal receptor-binding domain of humanIL-2. By insertion mutagenesis we have increased the apparentflexibility of the polypeptide chain between the membraneassociatingdomains and the receptor-binding domain of this fusion toxin.In comparison to DAB₄₈₆-IL-2, the cytotoxic potency of the insertionmutants was increased by 17-fold for high-affinity IL-2-receptor-bearingcell lines in vitro. Moreover, competitive displacement experimentsusing [¹²⁵I]rIL-2 demonstrate that the increase in cytotoxicpotency correlates with an increase in receptor-binding affinityfor both the high and intermediate forms of the IL-2 receptor.     

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.     

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue

Two residues, K89 and S380, thought to interact with the -carboxylgroup of the substrate L-glutamate, have been altered by site-directedmutagenesis of clostridial glutamate dehydrogenase (GDH). Thesingle mutants K89L and S380V and the combined double mutantK89L/S380V were constructed. All three mutants were satisfactorilyoverproduced in soluble form. However, only the K89L mutantwas retained by the dye column normally used in purifying thewild-type enzyme. All three mutant enzymes were purified tohomogeneity and tested for substrate specificity with 24 aminoacids. The single mutant S380V showed no detectable activity.The alternative single mutant K89L showed an activity towardsL-glutamate that was decreased nearly 2000-fold compared withwild-type enzyme, whereas the activities towards the monocarboxylicsubstrates -aminobutyrate and norvaline were increased 2- to3-fold. A similar level of activity was obtained with methionine(0.005 U/mg) and norleucine (0.012 U/mg), neither of which giveany activity with the wild-type enzyme under the same conditions.The double mutant showed decreased activity with all substratescompared with the wild-type GDH. In view of its novel activities,the K89L mutant was investigated in greater detail. A strictlylinear relationship between reaction velocity and substrateconcentration was observed up to 80 mM L-methionine and 200mM L-norleucine, implying very high K_m values. Values of k_cat/K_m,for L-methionine and L-norleucine were 6.7x10^–2 and 0.15s^–1M^–1, respectively. Measurements with dithiobisnitrobenzoicacid showed that the mutant enzymes all reacted with a stoichiometryof one -SH group per subunit and all showed protection by coenzyme,indicating essentially unimpaired coenzyme binding. With glutamateor 2-oxoglutarate as substrate the K_m values for the vestigialactivity in the mutant enzyme preparations were strikingly closeto the wild-type K_m values. Both for wild-type GDH and K89L,L-glutamate gave competitive product inhibition of 2-oxoglutaratereduction but did not inhibit the reduction of 2-oxocaproatecatalysed by K89L enzyme. This suggests that the low levelsof glutamate/2-oxoglutarate activity shown by the mutant enzymeare due to trace contamination. Since stringent precautionswere taken, it appears possible that this reflects the levelof reading error during overexpression of the mutant proteins.CD measurements indicate that the S380V mutant has an alteredconformation, whereas the K89L enzyme gave an identical CD spectrumto that of wild-type GDH; the spectrum of the double mutantwas similar, although somewhat altered in intensity. The resultsconfirm the key role of K89 in dicarboxylate recognition byGDH.     

Identification of two glutamic acid residues essential for catalysis in the {beta}-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus

The Sulfolobus solfataricus, strain MT4, ß-glycosidase(Ssßgly) is a thermophilic member of glycohydrolasefamily 1. To identify active-site residues, glutamic acids 206and 387 have been changed to isosteric glutamine by site-directedmutagenesis. Mutant proteins have been purified to homogeneityusing the Schistosoma japonicum glutathione S-transferase (GST)fusion system. The proteolytic cleavage of the chimeric proteinwith thrombin was only obtainable after the introduction ofa molecular spacer between the GST and the Ssß-glydomains. The Glu387 Gin mutant showed no detectable activity,as expected for the residue acting as the nucleophile of thereaction. The Glu206 Gin mutant showed 10- and 60-fold reducedactivities on aryl-galacto and aryl-glucosides, respectively,when compared with the wild type. Moreover, a significant K_mdecrease with plo-nitrophenyl-ß-D-glucoside was observed.The residual activity of the Glu206 Gln mutant lost the typicalpH dependence shown by the wild type. These data suggest thatGlu206 acts as the general acid/base catalyst in the hydrolysisreaction.     

Identification of two amino acids contributing the high enzyme activity in the alkaline pH range of an alkaline endoglucanase from a Bacillus sP

An alkaline cellulase ß-1,4-endoglucanase; NK1) froman alkalophilk Bacillus sp. shows great similarity in aminoacid sequence to a neutral cellulase (BSC) from Bacillus subtilis,despite a considerable difference in their pH activity profiles.Multiple amino acid exchanges by site-directed mutagenesis,using BSC as the reference, were performed on the residues inregion 5 of NK1, which was previously shown to be responsiblefor the high enzyme activity of this alkaline cellulase in abroad alkaline pH range. Two amino acid residues, Ser287 andAla296, were identified as being responsible for the activityin the alkaline range. The double mutation, Ser287 to Asn andAla296 to Ser, of NK1 made its pH activity profile almost thesame as that of BSC. On the other hand, the pH activity profilein the acidic range was not significantly affected by variousamino acid replacements including these two positions in region5. This observation, together with the information availableon other endoglucanases, suggests that the above two amino acidsubstitutions caused a profound effect through rearrangementof the hydrogen bond network forming the substrate-binding siteor the catalytic site.     

Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences

Site-directed mutagenesis was performed at Asp-Gly (48–49,66–67, 101–102) and Asn-Gly (103–104) sequencesof hen egg-white lysozyme to protect the enzyme against irreversiblethermoinactivation. Because the lysozyme inactivation was causedby the accumulation of multiple chemical reactions, includingthe isomerization of the Asp-Gly sequence and the deamidationof Asn [Tomizawa et al.(1994) Biochemistry, 33, 13032–13037],the suppression of these reactions by the substitution of Glyto Ala, or the introduction of a sequence of human-type lysozyme,was attempted and the mutants (where each or all labile sequenceswere replaced) were prepared. The substitution resulted in thereversible destabilization from 1 to 2 kcal/mol per substitution.The destabilization was caused by the introduction of ß-carbonto the constrained position that had conformational angles withinthe allowed range for the Gly residue. Despite the decreasein the reversible conformational stability, the mutants hadmore resistance to irreversible inactivation at pH 4 and 100°C.In particular, the rate of irreversible inactivation of themutant, which was replaced at four chemically labile sequences,was the latest and corresponded to 18 kcal/mol of the reversibleconformational stability. Therefore, replacement of the chemicallylabile sequence was found to be more effective at protectingenzymes against irreversible thermoinactivation than at strengtheningreversible conformational stability.     

Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver

We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 1654–1668, 1669–1682; 255,7291–7295, 7296–7300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (K_b, increasing it 5-fold from0.71 to 3.7 mM and reduced the k_cat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (K_ia) and Michaelis constant(K_a) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant.     

Cassette mutagenesis of Aspergillus awamori glucoamylase near its general acid residue to probe its catalytic and pH properties

Nine single amino add mutations in the active site of Aspergillusawamori glucoamylase were made by cassette mutagenesis to alterthe pH dependence of the enzyme and to determine possible functionsof the mutated residues. The Glul79-Asp mutation expressed inyeast led to a very large decrease in k_cat but to no changein K_m, verifying this residue's catalytic function. Aspl76-Gluand Glul80-Asp mutations affected K_m a more than k_cat, implyingthat Aspl76 and Glul80 are involved in substrate binding orstructural integrity. The Leul77-Asp mutation decreased k_catonly moderately, probably by changing the position of the generalacid catalytic group, and did not affect K_m. The Trpl78-Aspmutation greatly decreased k_cat while increasing K_m, showingthe importance of Trpl78 in the active site. Vall81-Asp andAsnl82-Asp mutations changed kinetk values little, suggestingthat Vall81 and Asnl82 are of minor catalytic and structuralimportance. Finally, insertions of Asp or Gly between residues176 and 177 resulted in almost complete loss of activity, probablycaused by destruction of the active site structure. No largechanges in pH dependence occurred in those mutations where kineticvalues could be determined, in spite of the increase in mostcases of the total negative charge. Increases in activationenergy of maltoheptaose hydrolysis in most of the mutant glucoamylasessuggested cleavage of individual hydrogen bonds in enzyme-substratecomplexes.