Asp Replacement Research Articles

From Alcohol Dehydrogenase to a “One-way” Carbonyl Reductase by Active-site Redesign: A MECHANISTIC STUDY OF MANNITOL 2-DEHYDROGENASE FROM PSEUDOMONAS FLUORESCENS

Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD(+)-mannitol (K(int)) was altered dramatically (10(4)- to 10(5)-fold) from being balanced in the wild-type enzyme (K(int) ≈ 3) to favoring enzyme-NAD(+)-mannitol in the single site mutants, N191D and N300D. The change in K(int) reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn --> Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD(+)-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn-->Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis.

The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter

EmrE is an Escherichia coli H(+)-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP(+), a high-affinity substrate, is 2 x 10(7) M(-1).s(-1), a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the k(off) of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs.

Stability Junction at a Common Mutation Site in the Collagenous Domain of the Mannose Binding Lectin

Missense mutations in the collagen triple-helix that replace one of the required Gly residues in the (Gly-Xaa-Yaa)(n)() repeating sequence have been implicated in various disorders. Although most hereditary collagen disorders are rare, a common occurrence of a Gly replacement mutation is found in the collagenous domain of mannose binding lectin (MBL). A Gly --> Asp mutation at position 54 in MBL is found at a frequency as high as 30% in certain populations and leads to increased susceptibility to infections. The structural and energetic consequences of this mutation are investigated by comparing a triple-helical peptide containing the N-terminal Gly-X-Y units of MBL with the homologous peptide containing the Gly to Asp replacement. The mutation leads to a loss of triple-helix content but only a small decrease in the stability of the triple-helix (DeltaT(m) approximately 2 degrees C) and no change in the calorimetric enthalpy. NMR studies on specifically labeled residues indicate the portion of the peptide C-terminal to residue 54 is in a highly ordered triple-helix in both peptides, while residues N-terminal to the mutation site have a weak triple-helical signal in the parent peptide and are completely disordered in the mutant peptide. These results suggest that the N-terminal triplet residues are contributing little to the stability of this peptide, a hypothesis confirmed by the stability and enthalpy of shorter peptides containing only the region C-terminal to the mutation site. The Gly to Asp replacement at position 54 in MBL occurs at the boundary of a highly stable triple-helix region and a very unstable sequence. The junctional position of this mutation minimizes its destabilizing effect, in contrast with the significant destabilization seen for Gly replacements in peptides modeling collagen diseases.

The Molecular Basis for the pH-activation Mechanism in the Channel-forming Bacterial Colicin E1

The in vitro activity of the channel-forming bacteriocins such as colicin E1 in model membranes requires the specific activation of the protein by an acidic environment in the presence of a membrane potential. Acid activation of the C-terminal domain results in the formation of an insertion-competent intermediate with an enhanced ability to penetrate and perforate cell membranes. We report novel findings of this activation process through the design and study of mutant proteins involving the replacement of conserved Asp residues Asp-408, Asp-410, and Asp-423 within helices 5a and 4 in the colicin E1 channel domain that resulted in enhanced membrane binding, bilayer insertion rates, and ion channel activities at near neutral pH values. This activation process involves the destabilization of a critical salt bridge (Asp-410 and Lys-406) and H-bonds (Asp-408 and Ser-405 main chain; Asp-423 and Lys-420 main chain). The helix-to-coil transition of this motif was identified previously by time-resolved Trp fluorescence measurements (Merrill, A. R., Steer, B. A., Prentice, G. A., Weller, M. J., and Szabo, A. G. (1997) Biochemistry 36, 6874-6884), and here we use this approach to demonstrate that disruption of the helical structure of helices 4 and 5a results in a shift in this equilibrium to favor the coil state. Finally, we show that the essential components of the pH trigger motif are conserved among the channel-forming colicins and that it likely exists within other bacterial proteins and may even have evolved into more sophisticated devices in a number of microbial species.

N‐terminal contributions of the γ‐subunit of fetal hemoglobin to its tetramer strength: Remote effects at subunit contacts

The greatly increased tetramer strength of liganded fetal hemoglobin compared with adult hemoglobin is shown by its 70-fold smaller tetramer-dimer dissociation constant. This property has been shown previously to be only partially caused by the 5-amino-acid differences at both types of interfaces in each hemoglobin. A major contributor to tetramer strengthening is the 18-amino-acid N-terminal A helix of the gamma-subunit of fetal hemoglobin, which differs from the beta-subunit of adult hemoglobin at eight amino acid residues. This long-distance communication between the A helix and the distant C helix and FG helical corner comprising the subunit contacts at the allosteric interface represents internal signaling. Physiologically, its greater tetramer strength endows fetal hemoglobin with the capacity to abstract oxygen from maternal adult hemoglobin. It also leads to resistance of fetal red cells to the malaria parasite because the HbF tetramer does not dissociate to dimers as readily as HbA; dimers are digested by malaria proteases but tetramers are not. In this communication, we report which sites on the A helix of the gamma-subunit are important for tetramer strengthening in HbF by substituting certain amino acids in the beta-subunit by the corresponding residues in the gamma-subunit. The recombinant hemoglobins containing up to five replacements together have been extensively characterized. Mass values were within 1 unit of theory. Gly 1 (gamma) of HbF with its high pK(a) of 8.1 compared with a 7.1 value for Val 1 (beta) of HbA creates a highly electropositive N terminus that may couple with the electronegative sequence just after it on the gamma-subunit. The Leu 3 to Phe replacement has no apparent role; however, position 5 is important because replacement of Pro 5 (beta) by Glu 5 (gamma) promotes tetramer strengthening. The Glu --> Asp replacement at position 7 enhances this effect because of the lower pK(a) of Asp but the Val --> Ile substitution at position 11 has no effect. Thus, the three positive/negative sites at positions 1, 5, and 7 account for practically all of the tetramer strength of HbF, as illustrated by an electrostatic surface potential analysis. The pathway by which information is transmitted to the distant allosteric subunit interfaces is currently under study. Oxygen-binding properties of the hemoglobins with charged substitutions more closely resemble those of HbA rather than those of HbF. Thus, whereas the A helix has a major role in controlling the strength of interactions at the tetramer-dimer allosteric interface, oxygen-binding properties of HbA and HbF are influenced by sequences in the C helix and at the FG helical corner constituting the allosteric interface.

Engineering the properties of a cold active enzyme through rational redesign of the active site

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.

Evidence for Interactions between Helices 5 and 8 and a Role for the Interdomain Loop in Tetracycline Resistance Mediated by Hybrid Tet Proteins

An interdomain hybrid Tet protein consisting of a class C alpha domain and a class B beta domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 microg/ml) compared with intact class B (256 microg/ml) and class C (64 microg/ml). Twenty-one independently isolated mutants of the Tet(C/B) protein with increased Tc resistance were generated by random chemical mutagenesis. Nine mutants with a Glu substitution for Gly-152 in helix 5 of the class C alpha domain produced a resistance of 48 microg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B beta domain mediated resistance at 32 microg/ml. The third type of mutation, found in 3 mutants expressing 24 microg/ml resistance, was a S202F replacement in the putative interdomain cytoplasmic loop of Tet(C/B). The latter underscores a previously unappreciated function of the interdomain cytoplasmic loop. All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored energy-dependent efflux of tetracycline. Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively charged side chain at position 152 was required for Tet(C/B) activity. These mutations appear to promote the necessary functional interactions between the interclass domains that do not occur in the Tet(C/B) hybrid protein and suggest a direct association between helix 5 and helix 8 in the function of Tet efflux proteins.

A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE.

EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent-solubilized protein binds tetraphenylphosphonium (TPP(+)) with a K(D) of 10 nM. One mole of ligand is bound per approximately 3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of approximately 7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane-embedded charged residue shows little transport activity, but binds TPP(+) at levels similar to those of the wild-type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.

The stability of holomyoglobin is determined by heme affinity.

The properties of wild-type, V68T, and H97D sperm whale myoglobins were compared to determine the relative importance of heme affinity and globin stability on the resistance of the holoprotein to denaturation. The V68T mutation decreases apoglobin stability by placing a polar side chain in the interior heme pocket. However, this substitution increases hemin affinity by formation of a strong hydrogen bond between coordinated water and the Thr68(E11) side chain. The H97D substitution disrupts favorable contacts with Ser92(F7) and the heme-7-propionate and causes a large increase in the rate of hemin dissociation. The Asp replacement has little affect on apoglobin stability because His97(FG3) is a surface residue. The aquomet, cyanomet, deoxyferrous, and apoglobin forms of each mutant and wild-type myoglobin were unfolded by titration with guanidinium chloride. Even though holomyoglobin denaturation involves the dissociation of heme and should be dependent on protein concentration, nonspecific heme binding to unfolded states makes the overall process appear to be a simple, unimolecular unfolding transition. The equilibrium constants for the denaturation of the holomyoglobin mutants correlate almost exclusively with heme affinity and not with the stability of the globin portion of the molecule. The strong correlation with heme affinity explains quantitatively why the stability of myoglobin is enhanced approximately 60-fold by reduction of iron to the ferrous deoxy state and by another approximately 100-fold with CO coordination. Parameters measured for GdmCl-, urea-, acid-, and heat-induced denaturation of holomyoglobins and hemoglobins reflect heme affinity and not the folding properties of the corresponding apoproteins. This conclusion suggests that (1) many previous studies of the denaturation of intact heme proteins need to be reevaluated in terms of heme affinity and (2) measurements with apoproteins are required for unambiguous determinations of the stability of globin structures.

β-γ Subunit Interaction Is Required for Catalysis by H+-ATPase (ATP Synthase): β SUBUNIT AMINO ACID REPLACEMENTS SUPPRESS A γ SUBUNIT MUTATION HAVING A LONG UNRELATED CARBOXYL TERMINUS

The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150-->Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.

Restoring allosterism with compensatory mutations in hemoglobin.

Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface. Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies.

Hb Fannin-Lubbock in five Spanish families is characterized by two mutations: beta 111 GTC-->CTC (Val-->Leu) and beta 119 GGC-->GAC (Gly-->Asp).

We have sequenced the amplified beta-globin genes of five, apparently unrelated, Spanish adults with a fast-moving hemoglobin variant, and observed a GGC-->GAC mutation at codon 119 which identified the abnormality as Hb Fannin-Lubbock or alpha 2 beta (2)119(GH2)Gly-->Asp. In addition, we found a GTC-->CTC change at codon 111 which leads to a Val-->Leu replacement at this location. Protein analysis of the beta A and beta X chains from one of these individuals confirmed that both mutations are located on the same chromosome. It is hypothesized that some other known variants may carry an additional mutation in one of their exons, resulting in a silent amino acid substitution which may have an effect on some physicochemical property. In the case of Hb Fannin-Lubbock, it appears likely that the Val-->Leu replacement at beta 111, rather than the Gly-->Asp replacement of beta 119, is the cause of the instability of the variant. The Hb Fannin-Lubbock variant in these Spanish families had a normal oxygen affinity.

Negative complementation in aspartate transcarbamylase. Analysis of hybrid enzyme molecules containing different arrangements of polypeptide chains from wild-type and inactive mutant catalytic subunits.

A comprehensive set of hybrid molecules of aspartate transcarbamylase (ATCase) from Escherichia coli has been constructed of wild-type and mutationally altered catalytic chains. The mutant enzymes that were virtually devoid of activity contained a replacement of Gly-128 in the catalytic polypeptide chains by either Asp or Arg. The kinetic properties of these hybrid enzyme-like molecules were analyzed to evaluate the basis for the unusual quaternary constraint demonstrated by an intersubunit hybrid containing one wild-type catalytic subunit, one inactive mutant subunit (containing the Gly to Asp replacement), and three wild-type regulatory subunits. A similar intersubunit hybrid was constructed from the wild-type catalytic subunit and the mutant in which Gly-128 was replaced by Arg, and it too demonstrated a pronounced decrease in activity relative to that expected for a hybrid containing three active sites. Moreover, neither of these hybrid holoenzymes exhibited the cooperativity with respect to aspartate that is characteristic of wild-type ATCase. In contrast, hybrid holoenzymes containing at least one wild-type chain in each catalytic subunit showed cooperativity. Also, hybrid enzymes containing different arrangements of five, four, three, or two wild-type catalytic chains with an appropriate complement of mutant chains had specific activities proportional to the number of wild-type chains in the holoenzymes. Exceptions were observed only in hybrids in which one of the two subunits in the holoenzyme was composed completely of mutant catalytic chains. For these hybrids the negative complementation was manifested as a much lower enzyme activity than expected from the number of wild-type chains in the enzyme and the loss of cooperativity. Thus, the activity and allosteric properties of these hybrids is dependent on the arrangement of catalytic chains in the holoenzyme, in contrast to results obtained for hybrids containing native and chemically modified catalytic chains. Intrasubunit hybrid catalytic trimers containing one or two wild-type chains exhibited one-third and two-thirds the activity of the intact wild-type catalytic subunit, respectively, indicating the dominant negative effect that was seen in intersubunit hybrid holoenzymes is absent within trimers.

Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression

Three different forms of dihydrofolate reductase (DHFR) from Escherichia coli with amino acid replacements Thr35----Asp, Asn37----Ser and Arg57----His, and one form containing all three of these changes were obtained by oligonucleotide-directed mutagenesis. These amino acids are on the surface of the protein and two of them (Thr35 and Arg57) are invariant for known sequences of DHFR. Conversion of Asn37----Ser has no effect on the functional activity or the protein level in the cells. The Thr35----Asp replacement leads to a sharp decrease in the protein level, while the addition of a DHFR inhibitor, trimethoprim (Tmp), to the growth medium increases the level of DHFR in the cells. There is a very small quantity of DHFR with all three amino acid changes. The addition of Tmp to the growth medium also leads to an increase in the mutant protein levels. The mutant with the Arg57----His replacement renders the cells sensitive to Tmp, but the level of DHFR is the same as for the wild-type protein. It is suggested that the invariant Thr35 is important for the stable conformation of DHFR whereas Arg57 is essential for protein activity. Various structural and functional aspects of these results are discussed.

Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli.

A 'split-gene' technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene. The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro followed by reconstitution in the pJLA504 expression vector under the transcriptional control of the lambda PR and lambda PL promoters and a temperature-sensitive lambda repressor. After thermo-induction, E. coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism. The strategy was used to engineer a Glu-188----Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties.

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285—-Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties.

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.

The complete amino-acid sequence of hen ovalbumin.

The complete amino acid sequence of hen ovalbumin, comprising 385 residues, has been determined. The sequence was deduced from the 17 cyanogen bromide fragments and from peptides derived by digestion with a number of proteolytic enzymes. The molecular weight of the polypeptide chain of ovalbumin is 42699. Ovalbumin has four sites of postsynthetic modification; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asn-292, and the two phosphorylated serines are at residues 68 and 344. The 'signal sequence' of ovalbumin is between residues 234 and 252. The heptapeptide released during the conversion of ovalbumin to plakalbumin by subtilisin digestion corresponds to residues 346-352. The hen ovalbumin polymorphism characterised by an Asn leads to Asp replacement results from a mutation at residue 311. The amino acid sequence of ovalbumin deduced from these amino acid sequence studies is in complete agreement with the sequence of mRNA determined by McReynolds et al. [Nature (Lond.) 273, 723-728 (1978)].