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Disruption of the Aldolase a Tetramer Into Catalytically Active Monomers PETER T

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Disruption of the Aldolase a Tetramer Into Catalytically Active Monomers PETER T Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5374-5379, May 1996 Biochemistry Disruption of the aldolase A tetramer into catalytically active monomers PETER T. BEERNINK* AND DEAN R. TOLANt Biology Department, Boston University, Boston, MA 02215 Communicated by Irwin A. Rose, Fox Chase Cancer Center, Philadelphia, PA, January 22, 1996 (received for review August 30, 1995) ABSTRACT The fructose-1,6-bisphosphate aldolase (EC interface loop of TIM resulted in a stable monomer with a 4.1.2.13) homotetramer has been destabilized by site-directed 103-fold reduction in kcat (11, 12). These studies have suggested mutagenesis at the two different subunit interfaces. A double that the quaternary structure is essential for proper catalytic mutant aldolase, Q125D/E224A, sediments as two distinct activity. species, characteristic of a slow equilibrium, with velocities Fructose-1,6-bisphosphate aldolase (Fru-1,6-P2; EC 4.1.2.13) is expected for the monomer and tetramer. The aldolase mono- an isologous homotetramer (Fig. 1A), each subunit ofwhich is an mer is shown to be catalytically active following isolation from eight-membered acq-barrel containing an active site near its sucrose density gradients. The isolated aldolase monomer had center (13). The two major subunit interfaces (A and B) are 72% of the specific activity of the wild-type enzyme and a distant (>20 A) from the Schiff base-forming Lys-229 (Fig. iB), slightly lower Michaelis constant, clearly indicating that the and aldolase does not exhibit cooperativity or allostery. Isolated quaternary structure is not required for catalysis. Cross- hybrids of isozymes indicated that subunits are catalytically linking of the isolated monomer confirmed that it does not independent; for example, A3B1 was indistinguishable from an rapidly reequilibrate with the tetramer following isolation. equivalent mixture of homotetramers, 3 A4: 1 B4 (14). Further- There was a substantial difference between the tetramer and more, hybrids of active and inactivated subunits also showed the monomer in their inactivation by urea. The stability toward independence of active sites (15). both urea and thermal inactivation of these oligomeric vari- Previous reports (16-18) indirectly suggested that mono- ants suggests a role for the quaternary structure in main- mers of rabbit Fru-1,6-P2 aldolase, isozyme A, exhibited taining the stability of aldolase, which may be an important substantial catalytic activity. Matrix-bound aldolase subunits role of quaternary structure in. many proteins. had 27% the specific activity of matrix-bound aldolase tetram- ers (17). Furthermore, the kinetics of reactivation during The quaternary structure, the three-dimensional arrangement refolding were biphasic, possibly indicating active assembly in- of subunits, has no known role in many proteins. Multimeric termediates (18). However, these experiments failed to demon- associations are critical for most biological functions; essential strate directly the association state(s) of the species examined. for the assembly of viral capsids, cytoskeletal structures, The stability and conservation of the aldolase tetramer are DNA-binding proteins, and cooperative regulatory enzymes; well documented. The dissociation constant of aldolase is and important in signal transduction pathways and multien- unmeasurable under nondenaturing conditions. The enzyme zyme complexes. Though the majority (70-80%) of enzymes did not observably dissociate at high dilution (0.2 ,ug/ml) in gel are oligomeric, only about 32% of oligomers exhibit cooper- filtration chromatography or in in vitro subunit exchange ativity (1). Therefore, the quaternary structure has some other experiments (19), nor was subunit exchange detected between function in most enzymes, such as forming the active site isozymes in vivo (20). Subunits from species as divergent as (directly or indirectly), providing stability, increasing solubil- chicken and wheat formed hybrid tetramers (21), indicating ity, or decreasing osmotic pressure in the cell (2, 3). that the subunit interfaces are functionally conserved among The role of quaternary structure can be examined by species. Although the quaternary structure is conserved, it has isolation of folded subunits by variation of physical conditions. no obvious structural or functional role. In many cases, dissociated subunits are inactive, possibly due As previously reported, substitutions of Asp-128, at the B to extreme conditions required to disrupt the enzyme or interface, disrupted the native quaternary structure, yielding because the subunits are inherently inactive. However, some active dimers, and reduced the thermal stability of the enzyme enzymes are regulated by association/dissociation, and in (22). Complete destabilization of the aldolase tetramer in- some cases subunits are more active than the oligomer (1). volved Carbamoyl-phosphate synthetase dissociation is promoted by construction of double mutant enzymes that combined dilution or by its allosteric activator, N-acetyl-L-glutamate, and one A and one B interface mutation. Polar or charged residues both dimeric and monomeric species are active (4). Malate that were invariant in a sequence alignment of vertebrate dehydrogenase dissociates as a function of Mg2+ concentration aldolases and were proximal to residues on an adjacent subunit and pH, and tetrameric, dimeric, and monomeric forms are were targeted. At the B interface, substitutions were made at active, with distinct kinetic parameters (5). The alcohol dehy- Asp-128 or Gln-125. Because the latter residue is proximal to drogenase 1313i dimer was disrupted using a freeze-thaw Asp-128 of the adjacent subunit, replacement with Asp may technique that resulted in active monomers (6). Thus it is clear create a charge repulsion. At the A interface, the charged atoms that at least some oligomeric enzymes are active as monomers. ofGlu-224 and Arg-258 ofadjacent subunits are <3.0A apart and More stable oligomeric proteins have been destabilized by putatively form four intersubunit ionic bonds per tetramer. site-directed mutagenesis, including triosephosphate isomer- Therefore, replacement of either of these residues was hypoth- ase (TIM) (7, 8), RecA (9), and prostatic acid phosphatase esized to destabilize the tetrameric structure. Single and double. (10). Although site-directed disruption of these enzymes re- sulted in complete loss of enzymatic activity, replacement of an Abbreviations: DMS, dimethyl suberimidate; Fru-1,6-P2, fructose-1,6- bisphosphate aldolase; TEA, triethanolamine; TIM, triosephosphate isomerase; G3PHD, glycerol-3-P dehydrogenase; MB, matrix bound. The publication costs of this article were defrayed in part by page charge *Present address: Department of Molecular and Cell Biology, Uni- payment. This article must therefore be hereby marked "advertisement" in versity of California, Berkeley, CA 94720. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5374 Downloaded by guest on September 25, 2021 Biochemistry: Beernink and Tolan Proc. Natl. Acad. Sci. USA 93 (1996) 5375 B A FIG. 1. Rabbit aldolase A crystal structure at 2.3-A resolution (13). (A) Aldolase tetramer with each subunit shown in a different color. The A interface is along the horizontal axis and the B interface is along the vertical axis. (B) One subunit is shown, with residues chosen for mutagenesis highlighted: Gln-125 (yellow), Asp-128 (violet), Glu-224 (light blue), and Arg-258 (red). The Schiff base-forming Lys-229 in the active site is shown in pink. interface mutants of aldolase were examined for catalytic prop- E224A double mutant was generated by using the E224A erties, thermal inactivation and quatemary structure. mutagenic oligonucleotide on template DNA carrying the Q125D mutation. The D128N/R258Q double mutant was constructed by cloning, replacing the 700-bp BsmI-HindIII MATERIALS AND METHODS fragment of pD128N with the corresponding fragment from Materials. Restriction endonucleases, T4 DNA ligase, exo- pR258Q. Potential mutants were screened by DNA sequence nuclease III, and DNA polymerase I were from New England determination using dideoxy termination (27). Biolabs. DNA polymerase I (Klenow fragment), calf intestine Expression and Purification of Recombinant Aldolase. The alkaline phosphatase, and glycerol-3-P dehydrogenase EcoRI-HindIII fragments containing the mutant genes were (G3PDH)/TIM were from Boehringer Mannheim. Deoxy- cloned into the high copy plasmid pPB1 (28) to create nucleoside triphosphates, Cm-Sepharose, CL-6B Fast Flow, pQ125D, pD128N, pE224A, pR258Q, and pQ125D/E224A. and Sephadex G-150 were from Pharmacia LKB. [a-35S]Deoxy- These plasmids were transformed into JM83 for protein nucleoside triphosphates were from Amersham. Nitrocellulose expression. The DNA sequence of the aldolase coding regions filters were from Sartorius. Oligonucleotides used for site- was determined to confirm that only the desired substitutions directed mutagenesis and sequencing were synthesized on were present. Restriction enzyme digestions, ligation reac- MilliGen/Biosearch DNA synthesizers using phosphoramidite tions, and transformations were performed as described (29). chemistry and the manufacturer's protocols. Ultrapure sucrose Wild-type and mutant aldolases were purified by existing was from GIBCO. Bio-lyte pH 3-10 ampholytes were from procedures (26), except that the Cm-Sepharose CL-6B Fast Bio-Rad. Fru-1,6-P2, dimethyl suberimidate (DMS), hydrazine flow column was washed with a buffer
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