Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.     

Circular permutation of 5-aminolevulinate synthase as a tool to evaluate folding, structure and function.

5-Aminolevulinate synthase, a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the condensation of glycine with succinyl-coenzyme A to yield aminolevulinate, carbon dioxide and CoA. This reaction corresponds to the first and regulatory step of the mammalian heme biosynthetic pathway. Mutations in the erythroid aminolevulinate synthase gene are associated with X-linked sideroblastic anemia, an erythropoietic disorder characterized by the presence of hypochromic-microcytic erythrocytes in peripheral blood and ring sideroblasts in bone marrow. In the past five years, transient kinetic studies in conjunction with three-dimensional structure models and engineered variants of aminolevulinate synthase have been instrumental in understanding the individual steps of the catalytic mechanism of aminolevulinate synthase. The mechanism of folding, assembly of the two subunits into a functional, dimeric holoenzyme has been recently explored in this laboratory using circular permutation of aminolevulinate synthase.     

Conversion of 5-aminolevulinate synthase into a more active enzyme by linking the two subunits: spectroscopic and kinetic properties

The two active sites of dimeric 5-aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are located on the subunit interface with contribution of essential amino acids from each subunit. Linking the two subunits into a single polypeptide chain dimer (2XALAS) yielded an enzyme with an approximate sevenfold greater turnover number than that of wild-type ALAS. Spectroscopic and kinetic properties of 2XALAS were investigated to explore the differences in the coenzyme structure and kinetic mechanism relative to those of wild-type ALAS that confer a more active enzyme. The absorption spectra of both ALAS and 2XALAS had maxima at 410 and 330 nm, with a greater A(410)/A(330) ratio at pH approximately 7.5 for 2XALAS. The 330 nm absorption band showed an intense fluorescence at 385 nm but not at 510 nm, indicating that the 330 nm absorption species is the substituted aldamine rather than the enolimine form of the Schiff base. The 385 nm emission intensity increased with increasing pH with a single pK of approximately 8.5 for both enzymes, and thus the 410 and 330 nm absorption species were attributed to the ketoenamine and substituted aldamine, respectively. Transient kinetic analysis of the formation and decay of the quinonoid intermediate EQ(2) indicated that, although their rates were similar in ALAS and 2XALAS, accumulation of this intermediate was greater in the 2XALAS-catalyzed reaction. Collectively, these results suggest that ketoenamine is the active form of the coenzyme and forms a more prominent coenzyme structure in 2XALAS than in ALAS at pH approximately 7.5.     

5-Aminolevulinate synthase and the first step of heme biosynthesis

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate. In animals, fungi, and some bacteria, 5-aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway. Mutations on the human erythroid 5-aminolevulinate synthase, which is localized on the X-chromosome, have been associated with X-linked sideroblastic anemia. Recent biochemical and molecular biological developments provide important insights into the structure and function of this enzyme. In animals, two aminolevulinate synthase genes, one housekeeping and one erythroid-specific, have been identified. In addition, the isolation of 5-aminolevulinate synthase genomic and cDNA clones have permitted the development of expression systems, which have tremendously increased the yields of purified enzyme, facilitating structural and functional studies. A lysine residue has been identified as the residue involved in the Schiff base linkage of the pyridoxal 5-phosphate cofactor, and the catalytic domain has been assigned to the C-terminus of the enzyme. A conserved glycine-rich motif, common to all aminolevulinate synthases, has been proposed to be at the pyridoxal 5phosphate-binding site. A heme-regulatory motif, present in the presequences of 5-aminolevulinate synthase precursors, has been shown to mediate the inhibition of the mitochondrial import of the precursor proteins in the presence of heme. Finally, the regulatory mechanisms, exerted by an iron-responsive element binding protein, during the translation of erythroid 5-aminolevulinate synthase mRNA, are discussed in relation to heme biosynthesis.     

Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation

FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined alpha-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1-10 microM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 degrees C of 15.2 kcal mol(-1) in urea and 13.5 kcal mol(-1) in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6 kcal mol(-1) in urea and 10.5 kcal mol(-1) in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, alpha-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, alpha-helical dimers.     

Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function

A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N:-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric. As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper folding, assembly, and stability of the holoenzyme.     

Dimer–Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA^Sec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA^Sec, formed by seryl-tRNA synthetase, to Sec-tRNA^Sec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA^Sec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA^Sec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.     

Random circular permutation of DsbA reveals segments that are essential for protein folding and stability

One of the key questions in protein folding is whether polypeptide chains require unique nucleation sites to fold to the native state. In order to identify possible essential polypeptide segments for folding, we have performed a complete circular permutation analysis of a protein in which the natural termini are in close proximity. As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues. To introduce new termini at all possible positions in its polypeptide chain, we generated a library of randomly circularly permuted dsbA genes and screened for active circularly permuted variants in vivo. A total of 51 different active variants were identified. The new termini were distributed over about 70 % of the polypeptide chain, with the majority of them occurring within regular secondary structures. New termini were not found in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA. Introduction of new termini into these "forbidden segments" by directed mutagenesis yielded proteins with altered overall folds and strongly reduced catalytic activities. In contrast, all active variants analysed so far show structural and catalytic properties comparable with those of DsbA wild-type. We suggest that random circular permutation allows identification of contiguous structural elements in a protein that are essential for folding and stability.     

Mechanism of 5-aminolevulinate synthase and the role of the protein environment in controlling the cofactor chemistry.

5-Aminolevulinate synthase, a pyridoxal 5'-phosphate-dependent enzyme of the alpha-oxoamine synthase family, catalyzes the first step of the heme biosynthetic pathway in mammalian cells. This reaction entails the condensation of glycine with succinyl-coenzyme A to yield 5-aminolevulinate, carbon dioxide and CoA. Mutations in the erythroid aminolevulinate synthase gene lead to a defective enzyme and are associated with the erythropoietic disorder X-linked sideroblastic anemia. In the past few years, rapid scanning-stopped-flow spectroscopy and chemical quenched-flow studies of the ALAS reaction, under single- and multi-turnover conditions, have provided important results for the interpretation of the catalytic mechanism. In particular, the role of the protein scaffold in modulating the chemical reactivity of the pyridoxal 5'-phosphate cofactor and, thus, the catalytic pathway of ALAS has been investigated in our laboratory using transient kinetics and global analysis of the kinetic data.     

Identification and characterization of key substructures involved in the early folding events of a (beta/alpha)8-barrel protein as studied by experimental and computational methods

A number of studies have examined the structural properties of late folding intermediates of (beta/alpha)8-barrel proteins involved in tryptophan biosynthesis, whereas there is little information available about the early folding events of these proteins. To identify the contiguous polypeptide segments important to the folding of the (beta/alpha)8-barrel protein Escherichia coli N-(5'-phosphoribosyl)anthranilate isomerase, we structurally characterized fragments and circularly permuted forms of the protein. We also simulated thermal unfolding of the protein using molecular dynamics. Our fragmentation experiments demonstrate that the isolated (beta/alpha)(1-4)beta5 fragment is almost as stable as the full-length protein. The far and near-UV CD spectra of this fragment are indicative of native-like secondary and tertiary structures. Structural analysis of the circularly permutated proteins shows that if the protein is cleaved within the two N-terminal betaalpha modules, the amount of secondary structure is unaffected, whereas, when cleaved within the central (beta/alpha)(3-4)beta5 segment, the protein simply cannot fold. An ensemble of the denatured structures produced by thermal unfolding simulations contains a persistent local structure comprised of beta3, beta4 and beta5. The presence of this three-stranded beta-barrel suggests that it may be an important early-stage folding intermediate. Interactions found in (beta/alpha)(3-4)beta5 may be essential for the early events of ePRAI folding if they provide a nucleation site that directs folding.     

Physico-chemical properties of molten dimer ascorbate oxidase

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 x 10(8) Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (DeltaV approximately -200 mL.mol(-1)) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (DeltaG approximately 2 kcal.mol(-1)) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.     

2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.     

Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release

5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.     

Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.     

Stereochemistry of the reactions of glutamate-1-semialdehyde aminomutase with 4,5-diaminovalerate

Conversion of glutamate 1-semialdehyde to the tetrapyrrole precursor, 5-aminolevulinate, takes place in an aminomutase-catalyzed reaction involving transformations at both the non-chiral C5 and the chiral C4 of the intermediate 4,5-diaminovalerate. Presented with racemic diaminovalerate and an excess of succinic semialdehyde, the enzyme catalyzes a transamination in which only the l-enantiomer is consumed. Simultaneously, equimolar 4-aminobutyrate and aminolevulinate are formed. The enzyme is also shown to transaminate aminolevulinate and 4-aminohexenoate to l-diaminovalerate as the exclusive amino product. The interaction of the enzyme with pure d- and l-enantiomers of diaminovalerate prepared by these reactions is described. Transamination of l-diaminovalerate yielded aminolevulinate quantitatively showing that reaction at the C5 amine does not occur significantly. A much slower transamination reaction was catalyzed with d-diaminovalerate as substrate. One product of this reaction, 4-aminobutyrate, was formed in the amount equal to that of the diaminovalerate consumed. Glutamate semialdehyde was deduced to be the other primary product and was also measured in significant amounts when a high concentration of the enzyme in its pyridoxal form was reacted with d-diaminovalerate in a single turnover. Single turnover reactions showed that both enantiomers of diaminovalerate converted the enzyme from its 420-nm absorbing pyridoxaldimine form to the 330-nm absorbing pyridoxamine via rapidly formed intermediates with different absorption spectra. The intermediate formed with l-DAVA (lambdamax = 420 nm) was deduced to be the protonated external aldimine with the 4-amino group. The intermediate formed with d-DAVA (lambdamax = 390 nm) was deduced to be the unprotonated external aldimine with the 5-amino group.     

Three-state kinetic folding mechanism of the H2A/H2B histone heterodimer: the N-terminal tails affect the transition state between a dimeric intermediate and the native dimer

The H2A/H2B heterodimer is a component of the nucleosome core particle, the fundamental repeating unit of chromatin in all eukaryotic cells. The kinetic folding mechanism for the H2A/H2B dimer has been determined from unfolding and refolding kinetics as a function of urea using stopped-flow, circular dichroism and fluorescence methods. The kinetic data are consistent with a three-state mechanism: two unfolded monomers associate to form a dimeric intermediate in the dead-time of the SF instrument (approximately 5 ms); this intermediate is then converted to the native dimer by a slower, first-order reaction. Analysis of the burst-phase amplitudes as a function of denaturant indicates that the dimeric kinetic intermediate possesses approximately 50% of the secondary structure and approximately 60% of the surface area burial of the native dimer. The stability of the dimeric intermediate is approximately 30% of that of the native dimer at the monomer concentrations employed in the SF experiments. Folding-to-unfolding double-jump experiments were performed to monitor the formation of the native dimer as a function of folding delay times. The double-jump data demonstrate that the dimeric intermediate is on-pathway and obligatory. Formation of a transient dimeric burst-phase intermediate has been observed in the kinetic mechanism of other intertwined, segment-swapped, alpha-helical, DNA-binding dimers, such as the H3-H4 histone dimer, Escherichia coli factor for inversion stimulation and E.coli Trp repressor. The common feature of a dimeric intermediate in these folding mechanisms suggests that this intermediate may accelerate protein folding, when compared to the folding of archael histones, which do not populate a transient dimeric species and fold more slowly.     

Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.     

Circular permutation of betaB2-crystallin changes the hierarchy of domain assembly.

The betagamma-crystallins form a superfamily of eye lens proteins comprised of multiple Greek motifs that are symmetrically organized into domains and higher assemblies. In the betaB2-crystallin dimer each polypeptide folds into two similar domains that are related to monomeric gamma-crystallin by domain swapping. The crystal structure of the circularly permuted two-domain betaB2 polypeptide shows that permutation converts intermolecular domain pairing into intramolecular pairing. However, the dimeric permuted protein is, in fact, half a native tetramer. This result shows how the sequential order of domains in multi-domain proteins can affect quaternary domain assembly.     

Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase.

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155' does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.     

Transient state kinetic investigation of 5-aminolevulinate synthase reaction mechanism

5-Aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the first, and regulatory, step of the heme biosynthetic pathway in nonplant eukaryotes and some bacteria. 5-Aminolevulinate synthase is a dimeric protein having an ordered kinetic mechanism with glycine binding before succinyl-CoA and with aminolevulinate release after CoA and carbon dioxide. Rapid scanning stopped-flow absorption spectrophotometry in conjunction with multiple turnover chemical quenched-flow kinetic analyses and a newly developed CoA detection method were used to examine the ALAS catalytic reaction and identify the rate-determining step. The reaction of glycine with ALAS follows a three-step kinetic process, ascribed to the formation of the Michaelis complex and the pyridoxal 5'-phosphate-glycine aldimine, followed by the abstraction of the glycine pro-R proton from the external aldimine. Significantly, the rate associated with this third step (k(3) = 0.002 s(-1)) is consistent with the rate determined for the ALAS-catalyzed removal of tritium from [2-(3)H(2)]glycine. Succinyl-CoA and acetoacetyl-CoA increased the rate of glycine proton removal approximately 250,000- and 10-fold, respectively, supporting our previous proposal that the physiological substrate, succinyl-CoA, promotes a protein conformational change, which accelerates the conversion of the external aldimine into the initial quinonoid intermediate (Hunter, G. A., and Ferreira, G. C. (1999) J. Biol. Chem. 274, 12222-12228). Rapid scanning stopped-flow and quenched-flow kinetic analyses of the ALAS reaction under single turnover conditions lend evidence for two quinonoid reaction intermediates and a model of the ALAS kinetic mechanism in which product release is at least the partially rate-limiting step. Finally, the carbonyl and carboxylate groups of 5-aminolevulinate play a major protein-interacting role by inducing a conformational change in ALAS and, thus, possibly modulating product release.