Site-directed mutagenesis identifies aspartate 33 as a previously unidentified critical residue in the catalytic mechanism of rabbit aldolase A (original) (raw)
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Subunit interface mutants of rabbit muscle aldolase form active dimers
Protein Science, 1994
We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase , Proc Natl Acud Sci USA 8436234627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase Dl280 purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wildtype rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes exist as mixtures of dimer and tetramer at 4 "C. Sedimentation at 20 "C shows that the mutant enzymes are >99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that T, values of the mutant enzymes are decreased by 12 "C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.
Subunit structure and chemical properties of rabbit liver aldolase
Journal of Biological Chemistry
A simple procedure for the isolation of large quantities of crystalline fructose diphosphate aldolase from rabbit livers is described. The enzyme is homogeneous as judged by its electrophoretic and sedimentation behavior. Molecular weight determinations by sedimentation analysis and gel filtration yield a value of 158,000. Upon treatment with guanidine hydrochloride or sodium dodecyl sulfate the enzyme dissociates into monomers of molecular weight of approximately 39,000, indicating a tetrameric quaternary structure.
The complete amino acid sequence of the human aldolase C isozyme derived from genomic clones
Biochimie, 1987
The complete protein sequence of the human aldolase C isozyme has been determined from recombinant genomic clones. A genomic fragment of 6673 base pairs was isolated and the DNA sequence determined. Aldolase protein sequences, being highly conserved, allowed the derivation of the sequence of this isozyme by comparison of open reading frames in the genomic DNA to the protein sequence of other human aldolase enzymes. The protein sequence of the third aldolase isozyme found in vertebrates, aldolase C, completes the primary structural determination for this family of isozymes. Overall, the aldolase C isozyme shared 81o70 amino acid homology with aldolase A and 70°?o homology with aldolase B. The comparisons with other aldolase isozymes revealed several aldolase C-specific residues which could be involved in its function in the brain. The data indicated that the gene structure of aldolase C is the same as other aldolase genes in birds and mammals, having nine exons sep~ated by eight introns, ~! in precisely the same positions, only the intron sizes being different. Eight of these exons contain the protein coding region comprised of 363 amino acids. The entire gene is approximately 4 kilobases.
Differential usage of the carboxyl-terminal region among aldolase isozymes
The Journal of biological chemistry, 1993
Sequence homology among nonconserved residues 357-362 of the COOH-terminal region in fructose-1,6-bisphosphate aldolases correlates with isozyme classification of aldolases. Recombinant chimers of human liver and maize aldolases were constructed by exchanging residues 357-362 with those from muscle, maize, and liver isozyme and by insertion in the maize sequence at position 349 rabbit muscle and liver residues 346-349. Activity variation among the chimers relative to native controls ranged from less than 10% to greater than 300% of Vm. Exchange of residues 357-362 significantly affected both Vm and Km without modifying catalytic efficiency kcat/Km, whereas insertion of residues 346-349 modified Vm and Km and increased catalytic efficiency. Steady state carbanion oxidation rates varied inversely with activity and were differentially affected with respect to equilibrium oxidation rates. Sequence exchange of residues 357-362 appears to modulate carbanion proton exchange, whereas sequen...
Complete amino acid sequence for human aldolase B derived from cDNA and genomic clones
Proceedings of the National Academy of Sciences, 1984
Several aldolase B clones from a human liver cDNA library have been identified by using a rabbit aldolase A cDNA as a hybridization probe. The most complete of these, pHL413, is 1389 base pairs long and covers -80% of the length of the mRNA, including 90% of the translated region. The cDNA, pHL413, was used to identify a genomic clone, XHG313, which encoded the remaining amino acids of human aldolase B. We demonstrate that the amino acid and nucleotide sequences of aldolase are strongly conserved even between different isozymes. Furthermore, in the 3'-untranslated regions of the mRNAs for the B isozyme of human and rat there is an extensive stretch of homology. Aldolase B lacks a cysteine at positions 72 and 338 and lacks a histidine at position 361. These residues, which are present in rabbit aldolase A, have previously been proposed to take part in catalysis. Our findings suggest that this may not be the case.
Biochemical Journal
Aldolase was purified from rabbit liver by affinity-elution chromatography. By taking precautions to avoid rupture of lysosomes during the isolation procedure, a stable form of liver aldolase was obtained. The stable form of the enzyme had a specific activity with respect to fructose 1,6-bisphosphate cleavage of 20-28,umol/min per mg of protein and a fructose 1,6-bisphosphate/fructose 1-phosphate activity ratio of 4. It was distinguishable from rabbit muscle aldolase, as previously isolated, on the basis of its electrophoretic mobility and N-terminal analysis. Muscle and liver aldolases were immunologically distinct. The stable liver aldolase was degraded with a lysosomal extract to a form with catalytic properties resembling those reported for aldolase B4. It is postulated that liver aldolase prepared by previously described methods has been modified by proteolysis and does not constitute the native form of the enzyme.
European Journal of Biochemistry, 1986
1. Brain-specific aldolase C amino acid sequence (> 75% of the coding region) was determined for the first time. Two cDNA clones, PAM1 and pAM2, were identified, from a mouse brain library, by using human aldolase B cDNA as a probe. The larger one, pAM2, identified as a cDNA for aldolase C, has been completely sequenced and covers the 5'-untranslated region of the mRNA and the codons for amino acids 1-227 of the protein. The sequence indicates that aldolase C is more akin to aldolase A than to aldolase B.