The catalytic properties of the pyruvate carboxylase enzyme were assessed follow

Question

The catalytic properties of the pyruvate carboxylase enzyme were assessed following purification. The activity of the enzyme was assayed in the presence of ATP, pyruvate, bicarbonate, and Mg2+ ions as a control. In addition, the dependence of the enzyme on these various metabolites was tested by replacing them with similar compounds. The activity of the enzyme in the presence of these various effectors is shown in Table 21.1.

Part A. Explain why PYC had no activity in the presence of avidin.

Part B. Is PYC dependent on ATP for activity? Can other nucleotides substitute for ATP? What is the

effect if other nucleotides are added to the assay mixture in addition to ATP?

Part C. What is the effect of other tricarboxylic acid metabolites on PYC activity in the methanogen?

Part D. What ion or ions are required for PYC activity?

As one of the reactants in the first reaction of the tricarboxylic acid cycle, oxaloacetate (OAA) is an important cellular metabolite. The concentrations of oxaloacetate are tightly regulated. Different organisms employ different mechanisms to obtain oxaloacetate. In mammals and yeast, OAA is the product of the pyruvate carboxylase (PYC) reaction. In E. coli, the enzyme phosphoeno pyruvate carboxylase (PPC) provides oxaloacetate from phosphoenolpyruvate obtained from glucose oxidation in the glycolytic pathway. If glucose is absent and E. coli is using acetate as a carbon source, the glyoxylate pathway serves to generate the needed oxaloacetate. Usually an organism will employ PPC or PYC, but not both. Detectable levels of PYC in the methanogenic bacterium Methanobacterium thermoautotrophicum had previously not been found, and since PPC had been detected, it was believed that M. thermoauto- trophicum did not possess PYC. However, in the case described here, microbiologists found that if they added biotin to cultures of the methanogenic bacterium, pyruvate carboxylase activity could be detected. This was a surprising finding, especially since it is known that the methanogen can synthesize its own biotin. However, having identified the presence of the PYC enzyme, the investigators set out to isolate, purify, and characterize the enzyme. Purification of the PYC was rather straightforward since the enzyme is soluble and hydrophilic. In addition, the investigators were able to make use of the protein avidin, which binds to biotin with high affinity and specificity

Explanation / Answer

A. The native protein structure of PYC is a tetramer, where the biotin carboxylase (BC), carboxyl transferase (CT), biotin-carboxyl carrier protein (BCCP) and allosteric (tetramerization ) domains are contained in a single polypeptide chain. Biotin, which is covalently attached to the -NH2 of a strictly conserved lysine residue located at the C-terminal end of the BCCP, is carboxylated at the N-1 position in the BC domain via the ATP-dependent activation of bicarbonate and formation of a putative carboxyphosphate intermediate. Carboxybiotin is required as a mobile carboxyl carrier, and gets translocated from the BC domain to a neighbouring CT domain to get decarboxylated. Therefore, biotin is required by PYC to effect the formation of oxaloacetate.

Avidin is a tetrameric protein (MW 63 kDa) which contains one biotin-binding site per subunit. The extremely high affinity of avidin for biotin makes it a potent inhibitor of biotin-dependent enzymes like PYC. Therefore, in the presence of avidin, PYC enzyme activity gets completely inhibited.

B.  The reaction catalysed in the BC domain, namely the
ATP-dependent carboxylation of biotin by bicarbonate is ATP dependent, Therefore, PYC activity is indeed dependent on ATP. Infact, MgATP is the physiological nucleotide substrate for the pyruvate carboxylation reaction of PYC. However, it cannot be substitued by other nucleotides since other nucleotides. Other nucleotides such as MgUTP, MgCTP, MgTTP and MgITP have been reported to act as competitive inhibitors of pyruvate carboxylation
with respect to MgATP. Without being complexed with Mg, these act as non-competitive inhibitors of PYC activity. Therefore, addition of these nucleotides in the assay mixture would result in reduced activity and inhibition of the reaction.

C. The ?4?4 PYCs (methanogen), unlike the ?4 enzymes (bacterial and eukaryotic), do not require acetyl-CoA for activity or stability and are insensitive to tricarboxylic acid cycle members or related metabolites. Thus, in respect to the quaternary structure and the requirement of or response to effectors, the PYC of some methanogens (M. thermoautotrophicum) is very mildly inhibited by ?-ketoglutarate. In the CT domain of the ?4 (bacterial and eukaryotic)PYC enzyme, carboxybiotin is decarboxylated. A strictly conserved Thr residue is proposed to facilitate the transfer of a proton from pyruvate to biotin, forming the highly nucleophilic enol-pyruvate. Pyruvate analogues and several dicarboxylic acids and their derivatives act as specific, reversible inhibitors of the carboxyl transfer step, through interactions with the Mn2+ - or Zn2+ -metal centre contained in the CT domain active site. Oxalate is also an effective inhibitor. Many of the derivatives of pyruvate, including fluoro and chloropyruvate, act as effective, specific inhibitors of the carboxyl transfer reaction. They function as carboxyl acceptors for the PYC-catalyzed carboxylation reaction. Also, aspartate, ?-ketoglutarate and glutamate are dicarboxylate allosteric inhibitors of PYC. Under physiological conditions, they provide regulatory feedback inhibition in response to the increased production of TCA cycle intermediates. Aspartate serves as an allosteric inhibitor of microbial and fungal PYC, while glutamate serves as inhibitor of PYC from vertebrate sources.

D. In the PYC-catalysed pyruvate carboxylation reaction two Mg2+ ions, are sequestered into the BC domain active site via the complexation to ATP. There is also a need for a divalent cation in the CT domain, generally Mn2+ or Zn2+, which is proposed to act as a Lewis acid and promote the enolization of pyruvate. Therefore, there appears to be an absolute mechanistic requirement of thse ions for PYC activity.

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