Enhancement of the Stability of a Prolipase from Rhizopus oryzae toward Aldehydes by Saturation Mutagenesis

A prolipase from Rhizopus oryzae (proROL) was engineered in order to increase its stability toward lipid oxidation products such as aldehydes with the aim of improving its performance in oleochemical industries. Out of 22 amino acid residues (15 Lys and 7 His) prone to react with aldehydes, 6 Lys and all His residues (except for the catalytic histidine) were chosen and subjected to saturation mutagenesis. In order to quickly and reliably identify stability mutants within the resulting libraries, active variants were prescreened by an activity staining method on agar plates. Active mutants were expressed in Escherichia coli Origami in a 96-well microtiterplate format, and a stability test using octanal as a model deactivating agent was performed. The most stable histidine mutant (H201S) conferred a stability increase of 60%, which was further enhanced to 100% by combination with a lysine mutant (H201S/K168I). This increase in stability was also confirmed for other aldehydes. Interestingly, the mutations did not affect specific activity, as this was still similar to the wild-type enzyme.     

Molecular studies of pH-dependent ligand interactions with the low-density lipoprotein receptor

The release of ligand from the low-density lipoprotein receptor (LDLR) has been postulated to involve a "histidine switch"-induced intramolecular rearrangement that discharges bound ligand. A recombinant soluble low-density lipoprotein receptor (sLDLR) was employed in ligand binding experiments with a fluorescently tagged variant apolipoprotein E N-terminal domain (apoE-NT). Binding was monitored as a function of fluorescence resonance energy transfer (FRET) from excited Trp residues in sLDLR to an extrinsic fluorophore covalently attached to Trp-null apoE3-NT. In binding experiments with wild-type (WT) sLDLR, FRET-dependent AEDANS fluorescence decreased as the pH was lowered. To investigate the role of His190, His562, and His586 in sLDLR in pH-dependent ligand binding and discharge, site-directed mutagenesis studies were performed. Compared to WT sLDLR, triple His --> Ala mutant sLDLR displayed attenuated pH-dependent ligand binding and a decreased level of ligand release as a function of low pH. When these His residues were substituted for Lys, the positively charged side chain of which does not ionize over this pH range, ligand binding was nearly abolished at all pH values. When sequential His to Lys mutants were examined, the evidence suggested that His562 and His586 function cooperatively. Whereas the sedimentation coefficient for WT sLDLR increased when the pH was reduced from 7 to 5, no such change occurred in the case of the triple Lys mutant receptor or a His562Lys/His586Lys double mutant receptor. The data support the existence of a cryptic, histidine side chain ionization-dependent alternative ligand that modulates ligand discharge via conformational reorganization.     

The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.     

Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.     

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.     

Sensor of phospholipids in Streptomyces phospholipase D

Recently, we identified Ala426 and Lys438 of phospholipase D from Streptomyces septatus TH-2 (TH-2PLD) as important residues for activity, stability and selectivity in transphosphatidylation. These residues are located in a C-terminal flexible loop separate from two catalytic HxKxxxxD motifs. To study the role of these residues in substrate recognition, we evaluated the affinities of inactive mutants, in which these residues were substituted with Phe and His, toward several phospholipids by SPR analysis. By substituting Ala426 and Lys438 with Phe and His, respectively, the inactive mutant showed a much stronger interaction with phosphatidylcholine and a weaker interaction with phosphatidylglycerol than the inactive TH-2PLD mutant. We demonstrated that Ala426 and Lys438 of TH-2PLD play a role in sensing the head group of phospholipids.     

Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor

The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process. We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cu(2+) ions. Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation. Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography. Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole. Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.     

Evidence for a proton transfer network and a required persulfide-bond-forming cysteine residue in Ni-containing carbon monoxide dehydrogenases

Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO(2) at a nickel-iron-sulfur active site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semiconserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. The activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or position 123. Activity was also rescued by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no electron paramagnetic resonance signals originating from the C-cluster. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or the stability of the C-cluster, and possibly for eliciting the redox chemistry of the C-cluster required for catalytic activity.     

Lys13 plays a crucial role in the functional adaptation of the thermophilic triose-phosphate isomerase from Bacillus stearothermophilus to high temperatures.

The thermophilic triose-phosphate isomerases (TIMs) of Bacillus stearothermophilus (bTIM) and Thermotoga maritima (tTIM) have been found to possess a His12-Lys13 pair instead of the Asn12-Gly13 pair normally present in mesophilic TIMs. His12 in bTIM was proposed to prevent deamidation at high temperature, while the precise role of Lys13 is unknown. To investigate the role of the His12 and Lys13 pair in the enzyme's thermoadaptation, we reintroduced the "mesophilic residues" Asn and Gly into both thermophilic TIMs. Neither double mutant displayed diminished structural stability, but the bTIM double mutant showed drastically reduced catalytic activity. No similar behavior was observed with the tTIM double mutant, suggesting that the presence of the His12 and Lys13 cannot be systematically correlated to thermoadaptation in TIMs. We determined the crystal structure of the bTIM double mutant complexed with 2-phosphoglycolate to 2.4-A resolution. A molecular dynamics simulation showed that upon substitution of Lys13 to Gly an increase of the flexibility of loop 1 is observed, causing an incorrect orientation of the catalytic Lys10. This suggests that Lys13 in bTIM plays a crucial role in the functional adaptation of this enzyme to high temperature. Analysis of bTIM single mutants supports this assumption.     

Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2alpha kinase

The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.     

Mutation of surface-exposed histidine residues of recombinant human granulocyte-colony stimulating factor (Cys17Ser) impacts on interaction with chelated metal ions and refolding in aqueous two-phase systems

Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.     

Differential effects of mutations in the chromophore pocket of recombinant phytochrome on chromoprotein assembly and Pr-to-Pfr photoconversion.

Site-directed mutagenesis was performed with the chromophore-bearing N-terminal domain of oat phytochrome A apoprotein (amino acid residues 1-595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore-binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore-binding and the Pr-->Pfr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild-type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild-type protein was found. The light-induced Pr-->Pfr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I700 decay and accelerated Pfr formation. P318 is also involved in the Pr-->Pfr conversion, the millisecond steps (formation of Pfr) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the Pr-->Pfr conversion with kinetics similar to wild-type protein.     

Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.     

Kinetic analysis of multiple proton shuttles in the active site of human carbonic anhydrase

We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.     

The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity.

Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.     

Active site residues in Mycobacterium tuberculosis pantothenate synthetase required in the formation and stabilization of the adenylate intermediate

Pantothenate synthetase (EC 6.3.2.1) catalyzes the formation of pantothenate from ATP, D-pantoate, and beta-alanine in bacteria, yeast, and plants. The three-dimensional structural determination of pantothenate synthetase from Mycobacterium tuberculosis has indicated specific roles for His44, His47, Asn69, Gln72, Lys160, and Gln164 residues in the binding of substrates and the pantoyl adenylate intermediate. To evaluate the functional roles of these strictly conserved residues, we constructed six Ala mutants and determined their catalytic properties. The substitution of alanine for H44, H47, N69, Q72, and K160 residues in M. tuberculosis pantothenate synthetase caused a greater than 1000-fold reduction in enzyme activity, while the Q164A mutant exhibited 50-fold less activity. The rate of the isolated adenylation reaction in single turnover studies was also reduced 40-1000-fold by the replacement of one of these six amino acids with alanine, suggesting that these residues are essential for the formation of the pantoyl adenylate intermediate. The rate of pantothenate formation from the adenylate and beta-alanine in the second half reaction could not be measured for the H44A, H47A, N69A, Q72A, and K160A mutants and was reduced 40-fold in the Q164A mutants. The activity of the K160C mutant enzyme was markedly enhanced by the alkylation of cysteine with bromoethylamine, further supporting the critical role of the K160 residue in pantoyl adenylate formation. Isothermal titration microcalorimetry analysis demonstrated that the substitution of either H47 or K160 for Ala resulted in a decreased affinity of the enzyme for ATP. These results indicate that the highly conserved His44, His47, Asn69, Gln72, Lys160 and residues are essential for the formation and stabilization of pantoyl adenylate intermediate in the pantothenate synthetase reaction.     

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.     

Conserved histidine residues in soybean lipoxygenase: functional consequences of their replacement.

Sequences of 13 lipoxygenases from various plant and mammalian species, thus far reported, display a motif of 38 amino acid residues which includes 5 conserved histidines and a 6th histidine about 160 residues downstream. These residues occur at positions 494, 499, 504, 522, 531, and 690 in soybean lipoxygenase isozyme L-1. Since the participation of iron in the lipoxygenase reaction has been established and existing evidence based on M?ssbauer and EXAFS spectroscopy suggests that histidines may be involved in iron binding, the effect of the above residues has been examined in soybean lipoxygenase L-1. Six singly mutated lipoxygenases have been produced in which each of the His residues has been replaced with glutamine. Two additional mutants have been constructed wherein the codons for His-494 and His-504 have been replaced by serine codons. All of the mutant lipoxygenases, which were obtained by expression in Escherichia coli, have mobilities identical to that of the wild-type enzyme on denaturing gel electrophoresis and respond to lipoxygenase antibodies. The mutated proteins H499Q, H504Q, H504S, and H690Q are virtually inactive, while H522Q has about 1% of the wild-type activity. H494Q, H494S, and H531Q are about 37%, 8%, and 20% as active as the wild type, respectively. His-517 is conserved in the several lipoxygenase isozymes but not in the animal isozymes. The mutant H517Q has about 33% of the wild-type activity. The inactive mutants, H499Q, H504Q, H504S, and H690Q, become insoluble when heated for 3 min at 65 degrees C, as does H522Q. The other mutants and the wild-type are stable under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations in the autoinhibitor site of the regulatory subunit of cAMP-dependent protein kinase I. Replacement of Ala-97 and Ser-99 interferes with reassociation with the catalytic subunit.

Each regulatory (R) subunit of cAMP-dependent protein kinase contains an autoinhibitor site that lies approximately 90-100 residues from the amino terminus. In order to study the importance of this autoinhibitor site in the type I R-subunit for interacting with the catalytic (C) subunit, recombinant techniques were used to replace Ala-97 with Gln, His, Lys, and Arg and to replace Ser-99 with Gly and Lys. All of the mutant proteins having a replacement at Ala-97 showed reduced affinity for the C-subunit ranging from 14- to 55-fold. In general, the decrease in affinity of the Ala-97 mutants for the C-subunit correlated with the increase in size of the side chain. In contrast to wild type R-subunit, where MgATP facilitates holoenzyme formation, MgATP inhibits the reassociation in all of the Ala-97 mutants suggesting that the larger side chains sterically interfere with bound MgATP in the active site of the C-subunit. Whereas MgATP slowed holoenzyme formation, AMP actually accelerated the reassociation of the A97K, A97H (pH 6.0), and A97Q mutants with the C-subunit. Therefore, the side chains of Lys-97, His-97, and Gln-97 can interact either electrostatically or by hydrogen bonding with the phosphate of AMP. This interpretation is reinforced by the fact that the stimulatory effect of AMP on the A97H mutant was pH-dependent. The affinities of the S99G and S99K mutants for the C-subunit were reduced 7- and 24-fold, respectively, suggesting that Ser-99 also may contribute to interactions between the R- and C-subunits.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices.

Three conserved aspartyl residues located in the putative transmembrane helices in the Tn10-encoded metal-tetracycline/H+ antiporter were replaced by Asn, Lys, or Glu with oligonucleotide-directed site-specific mutagenesis. Replacement of Asp84 or Asp15 by Asn or Lys caused a severe defect in tetracycline transport activity, however, the Glu84 and Glu15 mutants retained 150 and 40% of the wild type activity, respectively, indicating the critical role of the negative charge. The increase in the activity of the Glu84 mutant was due to an increase in the affinity for the substrate. H+/tetracycline coupling was intact in these mutants, including Asn and Lys mutants. On the other hand, all of the Asp285-substitution mutants showed a severe defect in tetracycline transport activity and a complete lack of tetracycline-coupled H+ transport. However, since in vivo tests showed the tetracycline resistance for the Glu285 mutant, a negative charge in position 285 plays some role in maintaining the possible down-hill and/or low affinity efflux of accumulated tetracycline from intact cells. Similar work was done for Asp365, and here the Asn and Glu mutants showed decreased but high activity, while the Lys mutant was only marginally active (5%), indicating that a negative charge is not so demanding in position 365, possibly because it is not in the membrane.