Catalytic Strategy of Citrate Synthase: Subunit Interactions Revealed as a Consequence of a Single Amino Acid Change in the Oxaloacetate Binding Site

Kurz, Linda C.; Shah, Saurabh; Frieden, Carl; Nakra, Tanuj; Stein, Richard E.; Drysdale, George R.; Evans, Claudia T.; Srere, Paul A.

doi:10.1021/bi00041a003

Cited by 25 publications

(35 citation statements)

References 26 publications

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“…The K m -values of CS4 for acetyl CoA and OAA were determined by keeping one substrate concentration constant (200 μM) and by varying the amount of the other (2.5-120 μM) ( Figure 2D). While the K m -values for acetyl CoA and OAA were with 16.5 and 49.4 μM, respectively, in the range of the K m -value observed for pea (31 and 16 μM) and tomato (18 and 19 μM), they were slightly higher than those observed for porcine mitochondrial citrate synthase (5 and 5.9 μM) (Iredale, 1979;Jeffery et al, 1988;Kurz et al, 1995). By comparing the K m -values as well as the specificity constants (k cat /K m ) for OAA and acetyl CoA, it becomes clear that CS4 has a higher affinity for acetyl CoA than OAA (Table 1).…”

Section: The Recombinant 6xhis-cs4 Protein Is An Active Citrate Synthmentioning

confidence: 66%

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

et al. 2014

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Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it catalyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intra- as well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.

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Section: The Recombinant 6xhis-cs4 Protein Is An Active Citrate Synthmentioning

confidence: 66%

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

et al. 2014

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“…Mutations in Cs gene can have a significant impact on catalytic properties of CS enzyme. For example, substitution of one amino acid in the active site for oxaloacetate binding produced a 600-fold decrease in catalytic activity of CS enzyme from the pig heart (18). The H55N substitution occurs in a proximity to the CS site interacting with acetyl CoA (27).…”

Section: Discussionmentioning

confidence: 99%

H55N polymorphism as a likely cause of variation in citrate synthase activity of mouse skeletal muscle

Ratkevičius

Carroll

Kilikevičius

et al. 2010

Physiological Genomics

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Citrate synthase (CS) is an enzyme of the Krebs cycle that plays a key role in mitochondrial metabolism. The aim of this study was to investigate the mechanisms underlying low activity of citrate synthase (CS) in A/J mice compared with other inbred strains of mice. Enzyme activity, protein content, and mRNA levels of CS were studied in the quadriceps muscles of A/J, BALB/cByJ, C57BL/6J, C3H/HeJ, DBA/2J, and PWD/PhJ strains of mice. Cytochrome c protein content was also measured. The results of the study indicate that A/J mice have a 50-65% reduction in CS activity compared with other strains despite similar levels of Cs mRNA and lack of differences in CS and cytochrome c protein content. CS from A/J mice also showed lower Michaelis constant (K(m)) for both acetyl CoA and oxaloacetate compared with the other strains of mice. In silico analysis of the genomic sequence identified a nonsynonymous single nucleotide polymorphism (SNP) (rs29358506, H55N) in Cs gene occurring near the site of the protein interacting with acetyl CoA. Allelic variants of the polymorphism segregated with the catalytic properties of CS enzyme among the strains. In summary, H55N polymorphism in Cs could be the underlying cause of low CS activity and its high affinity for substrates in A/J mice compared with other strains. This SNP might also play a role in resistance to obesity of A/J mice.

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“…The combined approaches of mutagenesis (13,22) and x-ray crystallography (23) have led to the assignment of porcine citrate synthase's Asp 375 as the general base involved in acetyl-CoA deprotonation and His 320 as the acid that protonates oxaloacetate's C-2 ketone. Elimination of either side chain produces a substantial diminution in condensation activity.…”

Section: Discussionmentioning

confidence: 99%

3-Hydroxy-3-methylglutaryl-CoA Synthase

Chun¹,

Vinarov²,

Zajíček³

et al. 2000

Journal of Biological Chemistry

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Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acylCoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by 13 C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu 95 functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK a of Glu 95 as required to support its function as a general acid.3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) 1 synthase catalyzes the committed step in ketogenic and cholesterogenic pathways. Early work (1, 2) on the purified enzyme identified reaction intermediates that indicate that catalysis of HMGCoA production involves a three-step process (Scheme 1).Recombinant forms of both the avian (3) and human (4) enzyme have been expressed in Escherichia coli and isolated at high levels of purity. The recombinant human enzyme has been used to demonstrate that HMG-CoA synthase is the target of a potent antisteroidogenic drug (4). Mutagenesis work on the recombinant avian enzyme (3) has confirmed that Cys 129 is required to form the acetyl-S-enzyme intermediate (Scheme 1), a hypothesis that was initially advanced on the basis of protein modification by a mechanism-based inhibitor (5, 6) as well as sequence analysis of a peptide that harbors the acetyl-S-Cys 129 adduct (7).2 Additionally, kinetic characterization of mutants in which His 264 has been replaced implicates that residue (8) in binding of the second substrate, acetoacetyl-CoA. The functions of other active site residues that participate directly in catalysis have not yet been firmly established.The sensitivity of HMG-CoA synthase activity to treatment with a carboxyl-directed modification reagent led to a preliminary observation (9), which implicated Glu 95 as a residue that is important to reaction chemistry. This report documents the crucial role of Glu 95 in catalysis and indicates which of the three steps in the reaction is compromised upon replacement of the Glu 95 carboxyl. Finally, potential functions for Glu 95 are considered, and tests ai...

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Catalytic Strategy of Citrate Synthase: Subunit Interactions Revealed as a Consequence of a Single Amino Acid Change in the Oxaloacetate Binding Site

Cited by 25 publications

References 26 publications

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

H55N polymorphism as a likely cause of variation in citrate synthase activity of mouse skeletal muscle

3-Hydroxy-3-methylglutaryl-CoA Synthase

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