Subunit structure of rabbit brain aldolase

Rabbit brain contains a mixture of aldolase A (muscle type) and aldolase C (brain type), present largely as the hybrid forms A₃C, A₂C₂, and AC₃, with smaller amounts of the homopolymers A₄ and C₄. We have developed new procedures for the isolation of the A-C hybrid set and the aldolase C subunits and compared the structure of these subunits with those of aldolase A. The two isoenzymes differ significantly in amino acid composition, but each contains three methionine residues per subunit and yields four peptides on cleavage with cyanogen bromide. The three methionine residues appear to occupy similar positions in the polypeptide chains but the molecular weight of the aldolase C subunit is only 37,000, approximately 10% smaller than that of the subunit of aldolase A. The difference is attributable to two or more deletions, totaling 30–40 amino acid residues, in two of the four BrCN peptides. The deletions include two of the buried cysteine residues that are located in the center of the polypeptide chain in aldolase A; these residues in aldolase A are, therefore, not involved in the contacts between the subunits in the tetramer. Aldolase C also lacks several of the histidine residues that are located near the active-site lysine residue of aldolase A, thus excluding these residues from participation in the catalytic mechanism.     

Catalytic activity of maize leaf phosphoenolpyruvate carboxylase in relation to oligomerization

The relationship between the state of oligomerization and activity of purified maize leaf phosphoenolpyruvate carboxylase using size exclusion high performance liquid chromatography was examined. Maximum activities of 35 to 38 micromoles per minute per milligram protein were found when 100% of the enzyme was in its tetrameric form. The effects of the sulfhydryl group modifiers CuCl₂ and p-chloromercuribenzoate on enzyme inhibition and the state of aggregation of the protein complex were examined. Aggregation of the enzyme is temperature and pH sensitive with low temperature and high pH favoring depolymerization. Stability of the tetrameric form is largely dependent upon histidyl residues, and to some extent this explains the biphasic response of enzyme activity to changes in MgCl₂ concentrations. Modification of the tetramer's histidyl residues by the inhibitor diethylpyrocarbonate (0.125 millimolar) results in its dissociation to the dimeric form and loss of activity. Subsequent treatment with 0.4 molar hydroxylamine results in reassociation to the tetramer and restoration of enzymic activity.     

Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.     

Guanidination of ovine luteinizing hormone and effects on activity.

The free amino groups in oLH, oLHalpha and oLHbeta were guanidinated by O-methylisourea. The epsilon-NH2 groups of lysine residues reacted bo substitute these positions in the sequence with the more basic homoarginine residue. The alpha-NH2 groups did not react under the conditions used. Guanidinated oLH or the products of guanidinated oLHalpha + native oLHbeta or guanidinated oLHalpha + guanidinated oLHbeta were inactive in two bioassay systems. Native oLHalpha + guanidinated oLHbeta, however, showed potencies of 39% to 55% of that observed with the native subunit recombinant or native oLH. Possible structural implications for hormone-receptor site interactions are discussed.     

Basis of thermostability in pig heart lactate dehydrogenase treated with O-methylisourea

Acetamidination of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with ethyl acetimidate resulted in an increase of thermostability, and covalent bridge formation between pairs of lysine residues is observed. Guanidination with O-methylisourea of the enzyme also increases the thermostability, but such a bridge seems not to be formed. Increased thermostability of guanidinated enzyme is considered to be due to the shift of the pK values of the lysine residues from 10.5 to 12.5 after guanidination. Modification experiments with carbodiimide reveals that the enzyme contains 4.6 pairs of neighboring lysine and carboxyl residues per subunit, and amide bonding between 3.2 pairs results in an increase of thermostability. Guanidination of 4.6 Lys/subunit of the enzyme yields an enzyme derivative with considerably increased thermostability. Salt bridge formation between the 4.6 pairs of neighboring carboxyl and guanidinated lysine residues per subunit might make a major contribution to the increased thermostability of the guanidinated enzyme.     

The NAD domain and the predicted structure for aldolase: a word of caution.

The proposal of E. Stellwagen [(1976) J. Mol Biol., 106, 903–911] that the structure of a protein can be predicted by sequence analysis provided that the protein specifically binds Cibacron blue F3GA, is not sound at least for muscle fructose bisphosphate aldolase. Contrary to the predictions we have shown that Cibacron blue does not interact directly with lysine 227 at the catalytic sites but with different sites which bind also ATP and fructose bisphosphate. We have shown also that aldolase binds 3.5 molecules of dye per subunit (dissociation constant 1.9 μm), too great a number to support the hypothesis that the binding of Cibacron blue is a specific indication of the presence of an NAD domain.     

Biosynthesis of tetrahydrofolate in plants: crystal structure of 7,8-dihydroneopterin aldolase from Arabidopsis thaliana reveals a novel adolase class

Dihydroneopterin aldolase (DHNA) catalyses a retroaldol reaction yielding 6-hydroxymethyl-7,8-dihydropterin, a biosynthetic precursor of the vitamin, tetrahydrofolate. The enzyme is a potential target for antimicrobial and anti-parasite chemotherapy. A gene specifying a dihydroneopterin aldolase from Arabidopsis thaliana was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity and crystallised using polyethylenglycol as the precipitating agent. The crystal structure was solved by X-ray diffraction analysis at 2.2 Å resolution. The enzyme forms a D₄-symmetric homooctamer. Each polypeptide chain is folded into a single domain comprising an antiparallel four-stranded β-sheet and two long α-helices. Four monomers are arranged in a tetrameric ring, and two of these rings form a hollow cylinder. Well defined purine derivatives are found at all eight topologically equivalent active sites. The subunit fold of the enzyme is related to substructures of dihydroneopterin triphosphate epimerase, GTP cyclohydrolase I, and pyruvoyltetrahydropterin synthase, which are all involved in the biosynthesis of pteridine type cofactors, and to urate oxidase, although some members of that superfamily have no detectable sequence similarity. Due to structural and mechanistical differences of DHNA in comparison with class I and class II aldolases, a new aldolase class is proposed.     

Fructose diphosphate aldolase from Mycobacterium smegmatis. Functional similarities with rabbit muscle aldolase.

Fructose diphosphate aldolase of Mycobacterium smegmatis is found to be a class I type aldolase and possesses functional similarities with rabbit muscle aldolase with respect to the amino acid residues at the catalytic site. The presence of a lysine residue at the active site is indicated by the formation of a Schiff-base with the substrate. The lower degree of inactivation compared to rabbit muscle aldolase on treatment with carboxypeptidase-A suggests the absence of an essential terminal tyrosine residue. Participation of histidine residues in enzyme catalysis is suggested by the photoinactivation of the enzyme in presence of methylene blue. Finally, thiol groups do not seem to have a direct role in catalysis.     

Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl₂ (2.1 Å; R = 18.2%, R_free = 21.9%), and in complex with glutamate, MgCl₂, and ADP (2.7 Å; R = 19.0%, R_free = 24.2%). Inspection of these structures reveals an unusual binding pocket for the α-carboxylate of the glutamate substrate and an ATP-independent Mg²⁺ coordination site, clarifying the Mg²⁺ dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.     

Oxalacetate decar☐ylase and pyruvate car☐ylase activities,and effect of sulfhydryl reagents in malic enzyme from Sulfolubus solfataricus

Malic enzyme (S)-malate: NADP⁺ oxidoreductase (oxaloacetate-decar☐ylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decar☐ylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decar☐ylation of l-malate. The oxaloacetate decar☐ylase activity was stimulated about 50% by NADP but only in the presence of MgCl₂, and was strongly inhibited by l-malate and NADPH which abolished the NADP activation. In the presence of MnCl₂ and in the absence of NADP, the Michaelis constant and V_m for oxaloacetate were 1.7 mM and 2.3 μmol·min⁻¹·mg⁻¹, respectively. When MgCl₂ replaced MnCl₂, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the K_m and V_m values for l-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5′-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decar☐ylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both l-malate and MnCl₂, and strongly enhanced by the car☐ylic acid substrates; NADP + malate + MnCl₂ afforded total protection. The inactivation of the oxaloacetate decar☐ylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decar☐ylase activity.     

Effects of Mg(2+) on activation of the (Na(+) + K(+)-dependent ATPase by Na(+1).

The effects of MgCl₂ on Na activation of three different enzymatic reactions catalyzed by a rat brain (Na + K)-dependent ATPase (adenosine 5′-triphosphatase) were studied. For the Na⁺-dependent ATPase reaction measured with 6 μm ATP, the K_0.5 for Na increased from 0.4 to 1.7 mm as the MgCl₂ concentration was raised from 50 to 2000 μm; the half-maximal effect occurred at a free Mg²⁺ concentration near 0.8 mm. By contrast, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na was again 0.4 mm, but further addition of 2 mm MgCl₂ then had little effect on the K_0.5 for Na. For the Na-dependent phosphorylation of the enzyme, measured with 6 μm ATP, the K_0.5 for Na increased similarly, from 0.2 to 0.8 mM, as the MgCl₂ concentration was raised from 50 to 2000 μm, but for the (Na + K)-dependent ATPase reaction the K_0.5 for Na was 13 mm and increased by only one-third as the MgCl₂ concentration was raised. The K_0.5 for K was also little affected by changes in MgCl₂ concentration. Finally, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na in the (Na + K)-dependent ATPase reaction decreased to 5 mm. These observations are considered in terms of an enzyme having high-affinity and low-affinity substrate sites, with occupancy of the low-affinity sites modifying Na activation differently, depending both on the specific reaction catalyzed and on whether occupancy is by free Mg²⁺ or by Mg-ATP.     

Making an Oriental equivalent of the yeast cytosolic aldehyde dehydrogenase as well as making one with positive cooperativity in coenzyme binding by mutations of glutamate 492 and arginine 480

Yeast has at least three partially characterized aldehyde dehydrogenases. Previous studies by gene disrupted in our laboratory revealed that the Saccharomyces cerevisiae cytosol ALDH1 played an important role in ethanol metabolism as did the class 2 mitochondrial enzyme. To date, few mutagenesis studies have been performed with the yeast enzymes. An important human variant of ALDH is one found in Asian People. In it, the glutamate at position 487 is replaced by a lysine. This glutamate interacts with an arginine (475) that is located in the subunit that makes up the dimer pair in the tetrameric enzyme. Sequence alignment shows that these two residues are located at positions 492 and 480, respectively, in the yeast class 1 enzyme which shares just 45% sequence identity with the human enzymes. Mutating glutamate 492 to lysine produced an enzyme with altered kinetic properties when compared to the wild-type glutamate-enzyme. The K_m for NADP of E492K increased to nearly 3600 μM compare to 40 μM for wild-type enzyme. The specific activity decreased more than 10-fold with respect to the recombinant wild-type yeast enzyme. Moreover, substituting a glutamine for a glutamate was not detrimental in that the E492Q had wild-type-like K_m for NADP and V_max. These properties were similar to the changes found with the human class 2 E487K mutant form. Further, mutating arginine 480 to glutamine produced an enzyme that exhibited positive cooperativity in NADP binding. The K_m for NADP increased 11-fold with a Hill coefficient of 1.6. The NADP-dependent activity of R480Q mutant was 60% of wild-type enzyme. Again, these results are very similar to what we recently showed to occur with the human enzyme [Biochemistry 39 (2000) 5295–5302]. These findings show that the even though the glutamate and arginine residues are not conserved, similar changes occur in both the human and the yeast enzyme when either is mutated.     

Stability of acetylated and superguanidinated chymotrypsinogens

Conversion of lysine residues to homoarginine led to protein stabilization as determined earlier by hydrogen isotope exchange (P. Cupo W. El-Deiry, P. L. Whitney and W. M. Awad, Jr., 1980, J. Biol. Chem.255, 10828–10833). In order to see if neutralization of charges on lysine residues affected stability, a homogeneous derivative of chymotrypsinogen was prepared wherein all amino groups were acetylated. Hydrogen isotope exchange studies indicated that the derivative was less stable than the native protein. In addition, highly guanidinated chymotrypsinogen was prepared by first coupling ethylenediamine to carboxyl groups of guanidinated chymotrypsinogen. Thereafter the protein was treated with O-methylisourea to form guanidinoethylamido groups at the ends of carboxyl residues. Acrylamide gel electrophoresis indicated that two products were formed. Hydrogen isotope exchange studies demonstrated that superguanidinated chymotrypsinogen is even less stable than the acetylated derivative. Thus guanidination of residues in addition to lysine does not lead to protein stabilization. The possibility is that such a highly cationic protein causes backbone fluctuations because of repulsion of surface charges.     

Properties of fructose 1,6-diphosphate aldolase from Drosophila melanogaster.

The amino acid composition and other properties of fructose 1,6-diphosphate aldolase from pupae of Drosophila melanogaster are reported and compared with those of other class I aldolases. Drosophila aldolase subunits contain only four residues of cysteine, five histidines, and two methionines. All four cysteine side chains react with 5,5′-dithiobis(2-nitrobenzoic acid) only in the presence of denaturating agent and are therefore thought to be buried within the molecule. With bromoacetate one carboxymethyl group is incorporated in the native enzyme with the loss of 90% of catalytic activity; inorganic phosphate is partially inhibiting this reaction. The near-uv absorption spectra of Drosophila and rabbit muscle aldolases are similar, the insect enzyme having higher absorbancies over the entire region corresponding to its higher tryptophan content. Circular dichroism-spectra of Drosophila aldolase indicate an α-helix content of 26%. Both the insect and vertebrate enzymes display marked tryptophan ellipticity bands between 290 and 300 nm.     

Studies on the structure of rabbit muscle aldolase. 3. Primary structure of the BrCN peptide containing the active site

The four peptide segments obtained from rabbit muscle aldolase by cleavage with BrCN and separation with gel-filtration chromatography (1) have been redesignated according to their positions in the molecule, N-A-B-C. The primary structure of segment A, containing 66 amino acid residues, including the Schiff base-forming lysine at the active site, has been elucidated by isolation and sequence analyses of the proteolytic subfragments. Preliminary separation of tryptic peptides containing 7–25 residues was achieved by chromatography on Sephadex G-25 which facilitated subsequent purification. For the study of the tryptic peptide of 25 residues further fragmentation with pepsin then subtilisin (Nagarse) was employed. Edman degradation directly after subtilisin cleavage of a peptide was found useful in avoiding deamidation of a glutamine NH₂-terminus newly formed in the proteolysis. The sequence of 90 amino acids in the center region of the polypeptide chain of rabbit muscle aldolase has now been established.     

Induction of mitochondrial contraction and concomitant inhibition of succinate oxidation by magnesium ions.

The addition of 1 Him MgCl₂ to partially swollen rat liver mitochondria respiring in an isoosmotic sucrose-sodium phosphate or sodium acetate medium containing 1 mm EDTA and 15 mm succinate (pH 7.2) initiates contraction, inhibits respiration, and alters the ultrastructural configuration of the inner membrane-cristae-matrix continuum. The maximal extent of contraction (A₅₂₀ increase) attainable with MgCl₂ in the phosphate medium is about 80% of the maximal contraction induced by 2,4-dinitrophenol but exceeds the maximal contraction induced with ADP by about 10%. The extent of mitochondrial contraction and the inhibition of succinate oxidation are dependent on MgCl₂ concentration. MgCl₂ at 1000 μm immediately inhibits about 70% of the succinate oxidation and initiates maximal extent of contraction concomitant with a distinct configurational change in ultrastructure which appears to differ from that initiated by either ADP or dinitrophenol. MgCl₂ at 150 μm does not inhibit the rate of succinate oxidation but it does initiate about 75% of the maximal contraction (A₅₂₀ increase) attained with 1000 μm MgCl₂. The rate of mitochondrial contraction is dependent on both the phosphate and MgCl₂ concentration. CaCl₂, in contrast to MgCl₂, immediately stimulates succinate oxidation and after a sharp contraction spike of short duration initiates additional expansion of the inner membrane-cristae-matrix continuum. The contraction spike occurs in a reaction system containing either phosphate or acetate. The results are consistent with the notion that Mg²⁺ and Ca²⁺ may modulate mitochondrial volume and exert control over certain oxidative processes.     

Location and primary structure of the substrate binding site peptide of aldolase C

An octadecapeptide containing the substrate-combining site of rabbit brain aldolase (aldolase C) has been isolated. This peptide has tentatively been assigned the structure: Ala-Leu-Ser(Asx,His,His,Val,Tyr)(Leu,Glx,Gly,Thr,Leu,Leu)(Lys,Pro,Asx,Met). The primary sequence of this peptide thus appears to be very similar to that of the active-site peptide of rabbit muscle aldolase (aldolase A), but it is located in a different BrCN segment, approximately 50 residues closer to the NH₂-terminus of the aldolase C subunit. A tentative sequence has also been obtained for an adjacent nonapeptide, also homologous with the corresponding structure in aldolase A. The evidence suggests that a large segment of the peptide chain in aldolase C may be translocated, as compared with aldolase A.     

Inactivation of phosphofructokinase by dialdehyde-ATP.

Rabbit muscle phosphofructokinase (PFK) is rapidly inactivated by a 2′,3′-dialdehyde derivative of adenosine triphosphate (dialdehyde-ATP). When allowed to react with 0.6 mm dialdehyde-ATP in 0.1 m borate buffer (pH 8.6) containing 0.2 mm EDTA and 0.5 mm dithiothreitol, PFK loses essentially all activity (99%) in 30 min. The modified PFK remains inactive following dialysis of the reaction mixture against sodium borate (pH 8.0) containing fructose diphosphate, EDTA, and dithiothreitol. Experiments with [¹⁴C]dialdehyde-ATP show that 99% inactivation of PFK corresponds to incorporation of 3 to 4 mol of the ATP analog per PFK protomer. The inactivation of PFK with dialdehyde reagent is not caused by dissociation of the 340,000 M_r, tetramer to the 170,000 M_r dimer, as determined by analytical ultracentrifugation. Adenosine diphosphate or ATP protect PFK from inactivation by dialdehyde-ATP at pH 8.6, but fructose 6-phosphate, cyclic 3′,5t$\text{-}\text{?}$adenosine monophosphate, or fructose diphosphate, which protect PFK from modification by pyridoxal phosphate, provide little protection from inactivation. Amino acid analyses of dialdehyde-inactivated PFK and of a control sample of the enzyme were compared following reaction of each with 2,4-dinitrofluorobenzene. The results show that three or four lysine residues per PFK protomer are modified by dialdehyde-ATP. Additional data indicate that these lysine residues react with dialdehyde-ATP to form dihydroxymorpholine-like adducts rather than Schiff bases.     

Intramolecular ionic interactions of lysine residues and a possible folding domain in fructose diphosphate aldolase.

1. Treatment with methyl acetimidate was used to probe the topography of the tetrameric fructose 1,6-diphosphate aldolase from ox liver. A single treatment with imido ester in the presence or absence of 20mM-fructose 1,6-diphosphate caused the number of amino groups in the enzyme to fall to approx. 30% of the starting number (assumed to be 30 per subunit). The catalytic activity of the aldolase modified in the presence of fructose 1,6-diphosphate was unaffected, whereas that of the enzyme modified in the absence of substrate fell by about 20%. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that the amino group of lysine-27 (the numbering is that of the highly homologous rabbit muscle enzyme) is essentially unavailable for amidination in the native enzyme and is therefore predicted to be buried in a hydrophobic environment, probably in the form of an ion-pair with a negatively charged side-chain carboxyl group. All the other lysine residues that reacted poorly with methyl acetimidate in the native enzyme (a total of 7) were found to be within the primary structure bounded by lysine-107 and lysine-227. An important member of this group of lysine residues displaying aberrant reactivity is lysine-227, which is known to form an imine with the substrate as part of the catalytic mechanism of the enzyme. 3. The results of the amidination experiments can be correlated in an interesting way with previous studies of thiol-group modification in the aldolases. Taken together, and arguing in part by analogy with the results of identical experiments with glyceraldehyde 3-phosphate dehydrogenases where the three-dimensional structure is known [Lambert & Perham (1977) Biochem. 4. 161. 49-62], they suggest that the region of primary structure from residues 107-227 may form the whole or part of a three-dimensional structural feature, perhaps a folding domain. A three-dimensional structure deduced from X-ray-crystallographic analysis will be needed to interpret these findings more closely. 4. The amino groups of lysine residues are commonly thought to reside at the 'surface' of protein structures. The patterns of specific lysine residues in glyceraldehyde 3-phosphate dehydrogenases and in aldolases that have been found to react poorly with methyl acetimidate in the native enzymes can be attributed to intramolecular ionic interactions deep in hydrophobic pockets and at the protein 'surface'. Such ionic interactions may contribute significantly to the stability of a given protein.