Role of the Calcium-Binding Residues Asp231, Asp233, and Asp438 in Alpha-Amylase of Bacillus amyloliquefaciens as Revealed by Mutational Analysis

Role of the calcium-binding residues Asp231, Asp233, and Asp438 of Bacillus amyloliquefaciens α-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233, and Asp438 were replaced with Asn, Asn, and Gly to produce the mutants D231N, D233N, and D438G, respectively. The mutant amylases were purified to homogeneity and the purified enzymes was estimated to be approximately 58 kDa. The specific activity for the mutant enzyme D233N was decreased by 84.8%, while D231N and D438G showed a decrease of 6.3% and 3.5% to that of the wild-type enzyme, respectively. No significant changes in the K _m value, thermo-stability, optimum temperature, and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (K _cat/K _m) and thermo-stability at 60°C. The ranges of optimum temperature and optimum pH for D233N were also reduced to about 10°C and 3–4 units, respectively.     

Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae

Site-directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae. Kinetic parameters for 4-methylumbelliferyl-N,N'-diacetyl-chitobiose or 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the Km values were different. The function of the carboxyl group of Asp313 was partially replaced by the amide of Asn when the 4-methylumbelliferyl-N,N',N"-triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.     

Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions.

The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->Asn and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, both in the presence of Cl- (150 mM) and in its absence (75 mM SO4(2-)). Under both conditions the color of the pigment is blue (lambda max = 615 nm). The time course of the photovoltage kinetics is similar to that of the acid-blue form of wild-type, except that an additional transient charge motion occurs with time constants of 60 microseconds and 1.3 ms, indicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is suggested that this is the group XH, which is responsible for proton release in wild-type. At pH 1, the photovoltage signal of D85N changes upon the addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore, the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pKa of the Schiff base, there is a mixture of "M-like" and "N-like" states. Net proton transport in the same direction as in wild-type is restored in D85N from this N-like state. With the mutant Asp212-->Asn (D212N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire pH range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage experiments indicate that net proton pumping is restored. However, this Cl(-)-induced activation of net charge-transport in D212N is only partial. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chloride-dependent features as those accompanying the acid-blue to acid-purple transition of the wild-type.     

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.     

Structural snapshots of MTA/AdoHcy nucleosidase along the reaction coordinate provide insights into enzyme and nucleoside flexibility during catalysis

MTA/AdoHcy nucleosidase (MTAN) irreversibly hydrolyzes the N9-C1' bond in the nucleosides, 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (AdoHcy) to form adenine and the corresponding thioribose. MTAN plays a vital role in metabolic pathways involving methionine recycling, biological methylation, polyamine biosynthesis, and quorum sensing. Crystal structures of a wild-type (WT) MTAN complexed with glycerol, and mutant-enzyme and mutant-product complexes have been determined at 2.0A, 2.0A, and 2.1A resolution, respectively. The WT MTAN-glycerol structure provides a purine-free model and in combination with the previously solved thioribose-free MTAN-ADE structure, we now have separate apo structures for both MTAN binding subsites. The purine and thioribose-free states reveal an extensive enzyme-immobilized water network in their respective binding subsites. The Asp197Asn MTAN-MTA and Glu12Gln MTAN-MTR.ADE structures are the first enzyme-substrate and enzyme-product complexes reported for MTAN, respectively. These structures provide representative snapshots along the reaction coordinate and allow insight into the conformational changes of the enzyme and the nucleoside substrate. A "catalytic movie" detailing substrate binding, catalysis, and product release is presented.     

Role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase: the amino acid residue which enhances the function of the enzyme-bound coenzyme pyridoxal 5'-phosphate.

Asp222 is an invariant residue in all known sequences of aspartate aminotransferases from a variety of sources and is located within a distance of strong ionic interaction with N(1) of the coenzyme, pyridoxal 5'-phosphate (PLP), or pyridoxamine 5'-phosphate (PMP). This residue of Escherichia coli aspartate aminotransferase was replaced by Ala, Asn, or Glu by site-directed mutagenesis. The PLP form of the mutant enzyme D222E showed pH-dependent spectral changes with a pKa value of 6.44 for the protonation of the internal aldimine bond, slightly lower than that (6.7) for the wild-type enzyme. In contrast, the internal aldimine bond in the D222A or D222N enzyme did not titrate over the pH range 5.3-9.5, and a 430-nm band attributed to the protonated aldimine persisted even at high pH. The binding affinity of the D222A and D222N enzymes for PMP decreased by 3 orders of magnitude as compared to that of the wild-type enzyme. Pre-steady-state half-transamination reactions of all the mutant enzymes with substrates exhibited anomalous progress curves comprising multiphasic exponential processes, which were accounted for by postulating several kinetically different enzyme species for both the PLP and PMP forms of each mutant enzyme. While the replacement of Asp222 by Glu yielded fairly active enzyme species, the replacement by Ala and Asn resulted in 8600- and 20,000-fold decreases, respectively, in the catalytic efficiency (kmax/Kd value for the most active species of each mutant enzyme) in the reactions of the PLP form with aspartate. In contrast, the catalytic efficiency of the PMP form of the D222A or D222N enzyme with 2-oxoglutarate was still retained at a level as high as 2-10% of that of the wild-type enzyme. The presteady-state reactions of these two mutant enzymes with [2-2H]aspartate revealed a deuterium isotope effect (kH/kD = 6.0) greater than that [kH/kD = 2.2; Kuramitsu, S., Hiromi, K., Hayashi, H., Morino, Y., & Kagamiyama, H. (1990) Biochemistry 29, 5469-5476] for the wild-type enzyme. These findings indicate that the presence of a negatively charged residue at position 222 is particularly critical for the withdrawal of the alpha-proton of the amino acid substrate and accelerates this rate-determining step by about 5 kcal.mol-1. Thus it is concluded that Asp222 serves as a protein ligand tethering the coenzyme in a productive mode within the active site and stabilizes the protonated N(1) of the coenzyme to strengthen the electron-withdrawing capacity of the coenzyme.     

Physicochemical characterisation of the two active site mutants Trp(52)-->Phe and Asp(55)-->Val of glucoamylase from Aspergillus niger

Glucoamylase 1 (GA1) from Aspergillus niger is a multidomain starch hydrolysing enzyme that consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated linker. The fungus also produces a truncated form without the starch-binding domain (GA2). The active site mutant Trp(52)-->Phe of both forms and the Asp(55)-->Val mutant of the GA1 form have been prepared and physicochemically characterised and compared to recombinant wild-type enzymes. The characterisation included substrate hydrolysis, inhibitor binding, denaturant stability, and thermal stability, and the consequences for the active site of glucoamylase are discussed. The circular dichroic (CD) spectra of the mutants were very similar to the wild-type enzymes, indicating that they have similar tertiary structures. The D55V GA1 mutant showed slower kinetics of hydrolysis of maltose and maltoheptaose with delta delta G(double dagger) congruent with 22 kJ mol(-1), whereas the binding of the strong inhibitor acarbose was greatly diminished by delta delta G degrees congruent with 52 kJ mol(-1). Both W52F mutant forms have almost the same stability as the wild-type enzyme, whereas the D55V GA1 mutant showed slight destabilisation both towards denaturant and heat (DSC). The difference between the CD unfolding curves recorded by near- and far-UV indicated that D55V GA1 unfolds through a molten globule intermediate.     

Deamidations in recombinant human phenylalanine hydroxylase. Identification of labile asparagine residues and functional characterization of Asn --> Asp mutant forms

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.     

Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin

Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D(140)XD(142)XE(144) sequence motif. In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k(cat) and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k(cat), indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue.     

Functional characterization of two variant human GSTO 1-1s (Ala140Asp and Thr217Asn)

Glutathione-S-transferase class Omega (GSTO 1-1) belongs to a new subfamily of GSTs, which is identical with human monomethylarsonic acid (MMA(V)) reductase, the rate limiting enzyme for biotransformation of inorganic arsenic, environmental carcinogen. Recombinant GSTO 1-1 variants (Ala140Asp and Thr217Asn) were functionally characterized using representative substrates. No significant difference was observed in GST activity towards 1-chloro-2,4-dinitrobenzene, whereas thioltransferase activity was decreased to 75% (Ala140Asp) and 40% (Thr217Asn) of the wild-type GSTO 1-1. For MMA(V) reductase activity, the Ala140Asp variant exhibited similar kinetics to wild type, while the Thr217Asn variant had lower V(max) (56%) and K(m) (64%) values than the wild-type enzyme. The different activities of the enzyme variants may influence both the intracellular thiol status and arsenic biotransformation. This can help explain the variation between individuals in their susceptibility to oxidative stress and inorganic arsenic.     

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.     

Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants

O-GlcNAcase is a family 84 beta-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of beta-O-linked 2-acetamido-2-deoxy-d-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants of human O-GlcNAcase, D174A and D175A, were generated since these residues are highly conserved among family 84 glycoside hydrolases. Structure-reactivity studies of the D174A mutant enzyme reveals severely impaired catalytic activity across a broad range of substrates alongside a pH-activity profile consistent with deletion of a key catalytic residue. The D175A mutant enzyme shows a significant decrease in catalytic efficiency with substrates bearing poor leaving groups (up to 3000-fold), while for substates bearing good leading groups the difference is much smaller (7-fold). This mutant enzyme also cleaves thioglycosides with essentially the same catalytic efficiency as the wild-type enzyme. As well, addition of azide as an exogenous nucleophile increases the activity of this enzyme toward a substrate bearing an excellent leaving group. Together, these results allow unambiguous assignment of Asp(174) as the residue that polarizes the 2-acetamido group for attack on the anomeric center and Asp(175) as the residue that functions as the general acid/base catalyst. Therefore, the family 84 glycoside hydrolases use a DD catalytic pair to effect catalysis.     

The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography

The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.     

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.     

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.     

Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis.

The proposed roles of Cys148 and Asp179 in deoxycytidylate (dCMP) hydroxymethylase (CH) have been tested using site-directed mutagenesis. CH catalyzes the formation of 5-(hydroxymethyl)-dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate. CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. Conversion of Cys148 to Asp, Gly, or Ser decreases CH activity at least 10(5)-fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys residue in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of the substrate dUMP to the side-chain amide of an Asn; the corresponding residue in CH is Asp179. Conversion of Asp179 to Asn reduces the value of kcat/KM for dCMP by (1.5 x 10(4))-fold and increases the value of kcat/KM for dUMP by 60-fold; as a result, CH(D179N) has a slight preference for dUMP. Wild-type CH and CH(D179N) are covalently inactivated by 5-fluoro-dUMP, a mechanism-based inactivator of TS. Asp179 is proposed to stabilize covalent catalytic intermediates, by protonating N3 of the pyrimidine-CH adduct.     

Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori

Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylase appeared by differential labeling to be in the active site. To test their functions, they were replaced by mutagenesis with Asn, Gln and Gln respectively, and kinetic parameters and pH dependencies of all enzyme forms were determined. Glu179----Gln glucoamylase was not active on maltose or isomaltose, while the kcat for maltoheptaose hydrolysis decreased almost 2000-fold and the KM was essentially unchanged from wild-type glucoamylase. The The Glu180----Gln mutation drastically increased the KM and moderately decreased the kcat with maltose and maltoheptaose, but affected isomaltose hydrolysis less. Difference in substrate activation energies between Glu180----Gln and wild-type glucoamylases indicate that Glu180 binds D-glucosyl residues in subsite 2. The Asp176----Asn substitution gave moderate increases and decreases in KM and kcat respectively, and therefore similar increases in activation energies for the three substrates. This and the differences in subsite binding energies between Asp176----Asn and wild-type glucoamylases suggest that Asp176 is near subsite 1, where it stabilizes the transition state and interacts with Trp120 at subsite 4. Glu179 and Asp176 are thus proposed as the general catalytic acid and base of pKa 5.9 and 2.7 respectively. The charged Glu180 contributes to the high pKa value of Glu179.     

Spontaneous chemical reversion of an active site mutation: deamidation of an asparagine residue replacing the catalytic aspartic acid of glutamate dehydrogenase

A mutant (D165N) of clostridial glutamate dehydrogenase (GDH) in which the catalytic Asp is replaced by Asn surprisingly showed a residual 2% of wild-type activity when purified after expression in Escherichia coli at 37 degrees C. This low-level activity also displayed Michaelis constants for substrates that were remarkably similar to those of the wild-type enzyme. Expression at 8 degrees C gave a mutant enzyme preparation 1000 times less active than the first preparation, but progressively, over 2 weeks' incubation at 37 degrees C in sealed vials, this enzyme regained 90% of the specific activity of wild type. This suggested that the mutant might undergo spontaneous deamidation. Mass spectrometric analysis of tryptic peptides derived from D165N samples treated in various ways showed (i) that the Asn is in place in D165N GDH freshly prepared at 8 degrees C; (ii) that there is a time-dependent reversion of this Asn to Asp over the 2-week incubation period; (iii) that detectable deamidation of other Asn residues, in Asn-Gly sequences, mainly occurred in sample workup rather than during the 2-week incubation; (iv) that there is no significant deamidation of other randomly chosen Asn residues in this mutant over the same period; and (v) that when the protein is denatured before incubation, no deamidation at Asn-165 is detectable. It appears that this deamidation depends on the residual catalytic machinery of the mutated GDH active site. A literature search indicates that this finding is not unique and that Asn may not be a suitable mutational replacement in the assessment of putative catalytic Asp residues by site-directed mutagenesis.     

Mutation of asparagine 229 to aspartate in thymidylate synthase converts the enzyme to a deoxycytidylate methylase.

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate dUMP. The Asn 229 to Asp mutant of Lactobacillus casei thymidylate synthase (TS N229D) has been prepared, purified, and investigated. Steady-state kinetic parameters of TS N229D show 3.5- and 10-fold increases in the Km values of CH2H4folate and dUMP, respectively, and a 1000-fold decrease in kcat. Most important, the Asp 229 mutation changes the substrate specificity of TS to an enzyme which recognizes and methylates dCMP in preference to dUMP. With TS N229D the Km for dCMP is bout 3-fold higher than for dUMP, and the Km for CH2H4folate is increased about 5-fold; however, the kcat for dCMP methylation is 120-fold higher than that for dUMP methylation. Specificity for dCMP versus dUMP, as measured by kcat/Km, changes from negligible with wild-type TS to about a 40-fold increase with TS N229D. TS N229D reacts with CH2H4folate and FdUMP or FdCMP to form ternary complexes which are analogous to the TS-FdUMP-CH2H4folate complex. From what is known of the mechanism and structure of TS, the dramatic change in substrate specificity of TS N229D is proposed to involve a hydrogen bond network between Asp 229 and the 3-N and 4-NH2 of the cytosine heterocycle, causing protonation of the 3-N and stabilization of a reactive imino tautomer. A similar mechanism is proposed for related enzymes which catalyze one-carbon transfers to cytosine heterocycles.     

Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin

Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D₁₄₀XD₁₄₂XE₁₄₄ sequence motif. In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k_cat and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k_cat, indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue.