Truncated Human ATP-Specific Succinyl-CoA Synthetase

Two isoforms of the heterodimeric enzyme succinyl-CoA synthetase (SCS) exist in the mitochondria of humans. One is specific for ATP, while the other is specific for GTP. Both catalyze the reversible reaction: succinate + CoA + NTP ⇌ succinyl-CoA + NDP + Pi, where N denotes adenosine or guanosine. SCS is best known as an enzyme of the citric acid cycle where the reaction generates NTP. In the reverse direction, SCS replenishes succinyl-CoA required for the catabolism of ketone bodies and for heme synthesis. Nucleotide-specific forms are thought to be required for SCS to serve its different metabolic roles. The nucleotide specificity lies in the β-subunit [1], and the β-subunit of human ATP-specific SCS has been shown to interact with the C-terminus of erythroid-specific aminolevulinic acid synthase (ALAS2) [2]. ALAS2 catalyzes the committed step in heme synthesis: succinyl-CoA + Gly ⇌ 5-aminolevulinate + CoA + CO₂. An interaction between SCS and ALAS2 makes biological sense, since this could provide channeling of succinyl-CoA from SCS to ALAS2. We hypothesize that the interaction is with the carboxy-terminus of the β-subunit of ATP-specific SCS because sequence comparisons show that the β-subunit of ATP-specific SCS has a carboxy-terminal extension when compared to other SCSs' β-subunits. To test this hypothesis, we added a carboxy-terminal His8-tag to the α-subunit of human ATP-specific SCS and mutated the codon for Thr 396β to a stop codon. This truncated version of human ATP-specific SCS has been produced in E. coli and purified. As well as testing to see if truncated human ATP-specific SCS interacts with ALAS2, we are using the truncated version in crystallization trials. Crystals of full-length human ATP-specific SCS diffract to only 3.2 Å and our goal is to obtain better-diffracting crystals of the complex of ATP with truncated human ATP-specific SCS.

Pig GTP-specific succinyl-CoA synthetase in complex with succinate

Succinyl-CoA synthetase (SCS) exists in the mitochondria of mammals as two different isoforms; one is ATP-specific and the other is GTP-specific. SCS is a heterodimer, and the two isoforms have a common α-subunit, but different β-subunits [1]. The β-subunit determines nucleotide specificity. Mutations in the α-subunit or the ATP-specific β-subunit can cause encephalomyopathy due to mitochondrial DNA depletion, along with lactic acidosis and methylmalonic aciduria (reviewed in [2]). The reaction catalyzed by SCS, succinyl-CoA+ NDP + Pi⇌succinate +CoA + NTP, is reversible, and the direction depends on the relative concentrations of substrates and products. Only after all substrate-binding sites are discovered can the catalytic mechanism of SCS be fully understood. Structures of SCS with ADP, GDP, GTP, Pi and CoA have been determined, but the succinate-binding site, or the binding site for the succinyl-portion of succinyl-CoA, is still unknown. Succinate is predicted to bind to the conserved sequence Gly-Gly-Ile-Val (327β-330β) located in a loop of the β-subunit of GTP-specific SCS. Crystals of other complexes with pig GTP-specific SCS have diffracted well, so we are crystallizing this enzyme in complex with succinate. Initially, plasmid containing the genes encoding pig GTP-specific SCS was transformed into E coli. After overproducing the desired protein with a 6-His tag on the C-terminus of the α-subunit, three different purification columns were used to obtain the GTP-SCS protein at high purity. Succinate was then co-crystallized with GTP-SCS under conditions containing polyethylene glycol 3350, magnesium formate and HEPES, pH 7.0.

Identification of Catalytic Residues in ATP-Citrate Lyase

An enzyme in the human body that regulates lipogenesis and cholesterolgenesis is ATP-citrate lyase (ACLY) [1]. ACLY synthesizes acetyl-CoA and oxaloacetate from citrate, CoA and ATP, with citryl-CoA as an intermediate [1]. The product acetyl-CoA is involved in cell growth as well as embryonic and brain development [1]. Studies in mice suggest that ACLY is important during brain development as homozygous Acly knockout mouse embryos died early in development [2]. Knowledge of the reaction mechanism is limited and will be of use in understanding the energy flow in cells. Our current insight into the mechanism by which citryl-CoA is cleaved to form acetyl-CoA and oxaloacetate is based on sequence similarity of ACLY to citrate synthase (CS). Both enzymes have histidine and aspartic acid residues at similar positions in their sequences. We hypothesize that citryl-CoA binds at this site in ACLY and is cleaved into acetyl-CoA and oxaloacetate using these residues. To test this hypothesis, we have mutated these residues to alanine in both human ACLY and ACLY from the bacterium, Chlorobium limicola. Enzymatic activities of the mutant proteins were tested using a coupled-enzyme assay with malate dehydrogenase. The inactive mutants are being used in crystallization trials with substrates, since complexes of the intermediate citryl-CoA can be trapped on the protein. To date, the crystal structure of full-length ACLY has not been published. The structure of only the amino-terminal two-thirds of the human enzyme has been determined [3]; however, the part of the protein that is similar to CS is the carboxy-terminal portion. This work identifies the catalytic residues of ACLY and complements the previous structure determination, increasing our currently limited knowledge about this enzyme.