Active site of 5-aminolevulinate synthase resides at the subunit interface. Evidence from in vivo heterodimer formation

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., et al. (1993) Protein Sci. 2, 1959-1965], while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function [Gong & Ferreira (1995) Biochemistry 34, 1678-1685]. To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants. When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E. coli strain acquired heme prototrophy. The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system. In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C. Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits.     

Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor

5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate. ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer. D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis. D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold. The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor. UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site. In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate. Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP. The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3). pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25. The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.     

Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase

Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.     

Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase

Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher Kd values for L-arginine (1-10 mM) than wild-type eNOS (0.4 microM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7, 8-tetrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (Kd ranged from 10 to 65 microM) except R372K. Heme spin-state changes caused by binding of 3, 5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.     

Role of lysine 39 of alanine racemase from Bacillus stearothermophilus that binds pyridoxal 5'-phosphate. Chemical rescue studies of Lys39 --> Ala mutant

The lysine residue binding with the cofactor pyridoxal 5'-phosphate (PLP) plays an important role in catalysis, such as in the transaldimination and abstraction of alpha-hydrogen from a substrate amino acid in PLP-dependent enzymes. We studied the role of Lys39 of alanine racemase (EC 5.1.1.1) from Bacillus stearothermophilus, the PLP-binding residue of the enzyme, by replacing it site-specifically with alanine and characterizing the resultant K39A mutant enzyme. The mutant enzyme turned out to be inherently inactive, but gained an activity as high as about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine. The amine-assisted activity of the mutant enzyme depended on the pKa values and molecular volumes of the alkylamines used. A strong kinetic isotope effect was observed when alpha-deuterated D-alanine was used as a substrate in the methylamine-assisted reaction, but little effect was observed using its antipode. In marked contrast, only L-enantiomer of alanine showed a solvent isotope effect in deuterium oxide in the methylamine-assisted reaction. These results suggest that methylamine serves as a base not only to abstract the alpha-hydrogen from D-alanine but also to transfer a proton from water to the alpha-position of the deprotonated (achiral) intermediate to form D-alanine. Therefore, the exogenous amine can be regarded as a functional group fully representing Lys39 of the wild-type enzyme. Lys39 of the wild-type enzyme probably acts as the base catalyst specific to the D-enantiomer of alanine. Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the K39A mutant.     

Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity

Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).     

Localization of the active site of type II dehydroquinases. Identification of a common arginine-containing motif in the two classes of dehydroquinases

A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have essential hyper-reactive arginine residues, and the type II enzymes have an essential tyrosine residue. The essential hyper-reactive Arg-23 of the Streptomyces coelicolor type II enzyme has been replaced by lysine, glutamine, and alanine residues. The mutant enzymes were purified and shown by CD spectroscopy to be structurally similar to the wild-type enzyme. All three mutant enzymes were much less active, for example the kcat of the R23A mutant was 30,000-fold reduced. The mutants all had reduced Km values, indicating stronger substrate binding, which was confirmed by isothermal titration calorimetry experiments. A role for Arg-23 in the stabilization of a carbanion intermediate is proposed. Comparison of the amino acid sequence around the hyper-reactive arginine residues of the two classes of enzymes indicates that there is a conserved structural motif that might reflect a common substrate binding fold at the active center of these two classes of enzyme.     

Bacterial selenocysteine synthase--structural and functional properties

Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.     

Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus

Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity. In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as natural toxin. Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins. These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25. NMR analyses (Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992) Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel alpha-helices, which together include 57% of the sequence. Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21. However, charge neutralizing mutations of the latter two sites have no effects on biological activity. Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.     

Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an approximately 2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.     

A new remote subsite in ribonuclease A

The interaction between bovine pancreatic ribonuclease A (RNase A) and its RNA substrate extends beyond the scissile bond. Enzymic subsites interact with the bases and the phosphoryl groups of a bound substrate. We evaluated the four cationic residues closest to known subsites for their abilities to interact with a bound nucleic acid. Lys-37, Arg-39, Arg-85, and Lys-104 were replaced individually by an alanine residue, and the resulting enzymes were assayed as catalysts of poly(cytidylic acid) (poly(C)) cleavage. The values of Km and kcat/Km for poly(C) cleavage were affected only by replacing Arg-85. Moreover, the contribution of Arg-85 to the binding of the ground state and the transition state was uniform---Km increased by 15-fold and kcat/Km decreased by 10-fold. The contribution of Arg-85 to binding was also apparent in the values of Kd for complexes with oligonucleotides of different length. This contribution was dependent on salt concentration, as expected from a coulombic interaction between a cationic side chain and an anionic phosphoryl group. Together, these data indicate that Arg-85 interacts with a particular phosphoryl group of a bound nucleic acid. We propose that Arg-85 comprises a new distal subsite in RNase A---the P(-1) subsite.     

Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II

Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.     

How enzymes control the reactivity of adenosylcobalamin: effect on coenzyme binding and catalysis of mutations in the conserved histidine-aspartate pair of glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through the formation of radical intermediates. It shares a structurally similar cobalamin-binding domain with methylcobalamin-dependent methionine synthase. In particular, both proteins contain the "DXHXXG" cobalamin-binding motif, in which the histidine provides the axial ligand to cobalt. The effects of mutating the conserved histidine and aspartate residues in methionine synthase have recently been described [Jarrett, J. T., Amaratunga, M., Drennan, C. L., Scholten, J. D., Sands, R. H., Ludwig, M. L., & Matthews, R. G. (1996) Biochemistry 35, 2464-2475]. Here, we describe how similar mutations in the "DXHXXG" motif of glutamate mutase affect coenzyme binding and catalysis in an adenosylcobalamin-dependent reaction. The mutations made in the MutS subunit of glutamate mutase were His16Gly, His16Gln, Asp14Asn, Asp14Glu, and Asp14Ala. All the mutations affect, in varying degrees, the rate of catalysis, the affinity of the protein for the coenzyme, and the coordination of cobalt. Mutations of either Asp14 or His16 decrease k(cat) by 1000-fold, and whereas cob(II)alamin accumulates as an intermediate in the wild-type enzyme, it does not accumulate in the mutants, suggesting the rate-determining step is altered. The apparent Kd for adenosylcobalamin is raised by about 50-fold when His16 is mutated and by 5-10-fold when Asp16 is mutated. There are extensive differences between the UV-visible spectra of wild-type and mutant holoenzymes, indicating that the mutant enzymes coordinate cobalt less well. Overall, the properties of these mutants differ quite markedly from those observed when similar mutations were introduced into methionine synthase.     

The primitive protozoon Trichomonas vaginalis contains two methionine gamma-lyase genes that encode members of the gamma-family of pyridoxal 5'-phosphate-dependent enzymes

Methionine gamma-lyase, the enzyme that catalyzes the breakdown of methionine by an alpha,gamma-elimination reaction and is a member of the gamma-family of pyridoxal 5'-phosphate-dependent enzymes, is present in high activity in the primitive protozoan parasite Trichomonas vaginalis but is absent from mammals. Two genes, mgl1 and mgl2, encoding methionine gamma-lyase, have now been isolated from T. vaginalis. They are both single copy, encode predicted proteins (MGL1 and MGL2) of 43 kDa, have 69% sequence identity with each other, and show a high degree of sequence identity to methionine gamma-lyase from Pseudomonas putida (44%) and other related pyridoxal 5'-phosphate-dependent enzymes such as human cystathionine gamma-lyase (42%) and Escherichia coli cystathionine beta-lyase (30%). mgl1 and mgl2 have been expressed in E. coli as a fusion with a six-histidine tag and the recombinant proteins (rMGL1 and rMGL2) purified by metal-chelate affinity chromatography. rMGL1 and rMGL2 were found to have high activity toward methionine (10.4 and 0.67 mumol/min/mg of protein, respectively), homocysteine (370 and 128 mumol/min/mg of protein), cysteine (6.02 and 1.06 mumol/min/mg of protein), and O-acetylserine (3.74 and 1.51 mumol/min/mg of protein), but to be inactive toward cystathionine. Site-directed mutagenesis of an active site cysteine (C113G for MGL1 and C116G for MGL2) reduced the activity of the recombinant enzymes toward both methionine and homocysteine by approximately 80% (rMGL1) and 90% (rMGL2). In contrast, the activity of mutated rMGL2 toward cysteine and O-acetylserine was increased (to 214 and 142%, respectively), whereas that of mutated rMGL1 was reduced to 39 and 49%, respectively. These findings demonstrate the importance of this cysteine residue in the alpha,beta-elimination and alpha, gamma-elimination reactions catalyzed by trichomonad methionine gamma-lyase.     

Prevalence and determinants of Helicobacter pylori infection in preschool children: a population-based study from Germany

Branching enzyme (BE) belongs to the amylolytic family which contains four highly conserved regions. These regions are proposed to play an important role in catalysis as they are thought to be necessary for catalysis and/or binding the substrate. Only one arginine residue was found to be conserved in a catalytic center at the same position in all known sequences of BEs from various species as well as in the alpha-amylase enzyme family. In mBEII, a conserved Arg residue 384 is in catalytic region 2. We have used site-directed mutagenesis of the Arg-384 residue in order to study its possible role in BE. Previous chemical modification studies (H. Cao and J. Preiss, 1996, J. Prot. Chem. 15, 291-304) suggest that it may play a role in substrate binding. Replacement of Arg-384 by Ala, Ser, Gln, and Glu in the active site caused almost total inactivation. However, a conservative mutation of the conserved Arg-384 by Lys resulted in some residual activity, approximately 5% of the wild-type enzyme. The kinetics of the purified mutant R384K enzyme were investigated and no large effect on the Km of the substrate amylose for BE was observed. Thus, these results suggest that conserved Arg residue 384 in mBEII plays an important role in the catalytic function of BEs but may not be directly involved in substrate binding.     

Role of specific amino acid residues in T4 endonuclease V that alter nontarget DNA binding

Endonuclease V is a pyrimidine dimer-specific DNA glycosylase-apurinic (AP)1 lyase which, in vivo or at low salt concentrations in vitro, binds nontarget DNA through electrostatic interactions and remains associated with that DNA until all dimers have been recognized and incised. On the basis of the analyses of previous mutants that effect this processive nicking activity, and the recently published cocrystal structure of a catalytically deficient endonuclease V with pyrimidine dimer-containing DNA [Vassylyev, D. G., et al. (1995) Cell 83, 773-782], four site-directed mutations were created, the mutant enzymes expressed in repair-deficient Escherichia coli, and the enzymes purified to homogeneity. Steady-state kinetic analyses revealed that one of the mutants, Q15R, maintained an efficiency (k(cat)/Km) near that of the wild-type enzyme, while R117N and K86N had a 5-10-fold reduction in efficiency and K121N was reduced almost 100-fold. In addition, K121N and K86N exhibited a 3-5-fold increase in Km, respectively. All the mutants experienced mild to severe reduction in catalytic activity (k(cat)), with K121N being the most severely affected (35-fold reduction). Two of the mutants, K86N and K121N, showed dramatic effects in their ability to scan nontarget DNA and processively incise at pyrimidine dimers in UV-irradiated DNA. These enzymes (K86N and K121N) appeared to utilize a distributive, three-dimensional search mechanism even at low salt concentrations. Q15R and R117N displayed somewhat diminished processive nicking activities relative to that of the wild-type enzyme. These results, combined with previous analyses of other mutant enzymes and the cocrystal structure, provide a detailed architecture of endonuclease V-nontarget DNA interactions.     

Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.     

Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes. Identification by complementary site-specific mutations

Ferredoxin reductase (Fd-reductase) supplies electrons to mitochondrial steroid hydroxylase cytochrome P450 enzymes via a [2Fe-2S] ferredoxin. Chemical labeling studies with bovine Fd-reductase have implicated Lys-243 as important in binding to bovine ferredoxin (Hamamoto, I., Kazutaka, K., Tanaka, S., and Ichikawa, Y. (1988) Biochim. Biophys. Acta 953, 207-213). We have used site-directed mutagenesis to examine the role of charged residues in this region of human Fd-reductase in ferredoxin binding. Mutant proteins were expressed in Escherichia coli and were assayed for activity by ferredoxin-mediated electron transfer to cytochrome c. Replacement of Lys-242 (homologous to Lys-243 in bovine Fd-reductase) with Gln and replacement of Arg-241 with Ser had little effect (2.7- and 3.6-fold increased Km, respectively). In contrast, mutants at positions 239 and 243 (R239S and R243Q) exhibited markedly lower affinity for ferredoxin (17.5- and 1,600-fold increased Km, respectively). Studies were also carried out with two ferredoxin charge mutants shown previously to have lowered affinity for Fd-reductase (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Comparisons of the binding of ferredoxin mutants D76N and D79N to Fd-reductase mutants R239S and R243Q suggest that Arg-239 and Arg-243 of Fd-reductase each interact directly with both Asp-76 and Asp-79 of ferredoxin during formation of the complex between the two proteins. These results support the hypothesis that specific electrostatic interactions involving this region are important in stabilizing the ferredoxin-Fd-reductase complex.     

Recombinant iron-regulatory factor functions as an iron-responsive-element-binding protein, a translational repressor and an aconitase. A functional assay for translational repression and direct demonstration of the iron switch

The translation of ferritin and erythroid 5-aminolevulinate synthase mRNAs is regulated via a specific high-affinity interaction between an iron-responsive element in the 5' untranslated region of ferritin and erythroid 5-aminolevulinate synthase mRNAs and a 98-kDa cytoplasmic protein, the iron-regulatory factor. Iron-regulatory factor was expressed in vaccinia-virus-infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N-terminal histidine tag allowed a rapid one-step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to iron-responsive elements and were immunoprecipitated by iron-regulatory-factor antibodies. Using in-vitro-transcribed chloramphenicol-acetyltransferase mRNAs bearing an iron-responsive element in the 5' untranslated region, specific repression of chloramphenicol-acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat-germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in iron-responsive-element-binding of iron-regulatory factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant iron-regulatory factor is a bifunctional protein; after purification, it binds to iron-responsive elements and represses translation in vitro. Following iron treatment, iron-responsive-element binding is lost and aconitase activity is gained. No eukaryotic co-factor seems to be required for the conversion of the iron-responsive-element binding to the aconitase form of the protein.     

Mutation of an active site residue of tryptophan synthase (beta-serine 377) alters cofactor chemistry

To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta-Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa approximately 7.7) from a form with a protonated internal aldimine nitrogen (lambdamax = 416 nm) to a deprotonated form (lambdamax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta2 subunit and alpha2 beta2 complex are pH-independent. The reaction of the S377D alpha2 beta2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambdamax = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.