Pyruvate enzyme active site

Step 1 of Figure 29.13 Carboxylation Gluconeogenesis begins with the carboxyl-afion of pyruvate to yield oxaloacetate. The reaction is catalyzed by pyruvate carboxylase and requires ATP, bicarbonate ion, and the coenzyme biotin, which acts as a carrier to transport CO2 to the enzyme active site. The mechanism is analogous to that of step 3 in fatty-acid biosynthesis (Figure 29.6), in which acetyl CoA is carboxylated to yield malonyl CoA. 

Pyruvate carboxylase has four identical subunits, each containing a molecule of biotin covalently attached through an amide linkage to the -amino group of a specific Lys residue in the enzyme active site. Carboxylation of pyruvate proceeds in two steps (Fig. 16-16) first, a carboxyl group derived from HCO3 is attached to biotin,... 

An understanding of mechanisms of thiamin pyrophosphate-dependent processes must begin with the classic work of Breslow 105, 134), who showed that the hydrogen at C-2 of thiamin pyrophosphate can be removed by bases and that the resulting anion is highly reactive. The at this site is 18 135). In an enzyme active site, this p a value may be considerably lower, as the value decreases with decreasing medium polarity. Reaction of the anion with a variety of carbonyl compounds (e.g., acetaldehyde, pyruvate) gives rise to characterizable adducts 132). 

As in the case of pyridoxal phosphate, the key to reaction in this case is the use of a heterocyclic compound as an electron sink in the decarboxylation step. Conformational control of the TPP-pyruvate adduct may also be important. The enzyme active site is probably nonpolar, and this provides a significant catalytic factor (112). 

Removal of CO2 from pyruvate. This reaction is carried out by the pyruvate decarboxylase (El) component of the complex. Like yeast pyruvate decarboxylase, responsible for the production of acetaldehyde, the enzyme uses a thiamine pyrophosphate cofactor and oxidizes the carboxy group of pyruvate to CO2. Unlike the glycolytic enzyme, acetaldehyde is not released from the enzyme along with CO2. Instead, the acetaldehyde is kept in the enzyme active site, where it is transferred to Coenzyme A. 

Neidig, M.L. et al. (2005). Spectroscopic and computational studies of NTBC bound to the non-heme iron enzyme (4-hydroxyphenyl) pyruvate dioxygenase active site contributions to drug inhibition. Biochem. Biophys. Res. Commun. 338, 206-214... 

The pyruvate dehydrogenase complex (PDC) is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. The active sites of ail three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme and so on, without diffusion of substrates and products through the solution. The overall reaction (see A Deeper Look Reaction Mechanism of the Pyruvate Dehydrogenase Complex ) involves a total of five coenzymes thiamine pyrophosphate, coenzyme A, lipoic acid, NAD+, and FAD. 

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (NAD nicotinamide adenine dinucleotide FAD, flavin adenine dinucleotide TDP, thiamin diphosphate.)...

Certain enzymes catalyze their reactions by way of a multisite mechanism in which the covalently linked intermediate is attached to a long arm that swings from one subsite to another subsite within the enzyme. In some cases, the covalently tethered intermediate can actually be transferred between subunits that form the active site. An example is Propionibacterium shermanii transcarboxylase an enzyme that catalyzes the biotin-dependent conversion of methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate. Carboxylated biotin allows the two catalytic subsites to operate on the same reaction intermediate. 

Nucleotide Base Conformation. Using NMR data, a relationship between the degree of specificity and the conformation of bound ATP at the active site has been shown for a number of ATP utilizing enzymes. Two examples of these are cAMP-dependent protein kinase and pyruvate kinase (18,19) It appears that enzymes that exhibit higher nucleotide triphosphate specificity bind ATP so... 

Affinity Labeling of Catalytic ATP Sites. Residues involved in ATP binding are potentially revealed by the use of affinity labels that are based on ATP s structure. Perhaps the most systematically studied of these compounds is 5 -fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been reported to label at least six kinases (32-A1). In the case of rabbit muscle pyruvate kinase such work has Indicated the presence of a tyrosine residue within the metal nucleotide binding site and an essential cysteine residue located at or near the free metal binding site (32). A similar reagent, 5 -FSBGuanosine, revealed the presence of two cysteine residues at the catalytic site of this same enzyme, both distinct residues from those modified by 5 -FSBA (33,34). With yeast pyruvate kinase both tyrosine and cysteine residues were modified by 5 -FSBA at the catalytic site ( ), and with porcine cAMP-dependent protein kinase a lysine residue was labeled at the active site (36). 

Since the discovery that glycolate was an alternate substrate for pyruvate kinase ( ), several other o-hydroxy acids have also been found to be substrates for this enzyme ( ). This class of alternate substrates provides a new approach the problem of substrate specificity for pyruvate kinase. 3-Nitrolactate is one such alternate substrate. Interestingly, the phosphorylated product of this reaction inactivates the enzyme (86). However, 3-nitrolac-tate does not behave as a straightforward affinity label since covalent modification occurs nonspecifically. It is hoped that this new Information may lead to the design of an affinity label of this enzyme, further serving to pinpoint amino acid groups at the active site. 

Fig. 9. A schematic drawing of a possible mechanism for the reaction catalyzed by the pyruvate dehydrogenase complex. The three enzymes Elf E2, and E3 are located so that lipoic acid covalently linked to E2 can rotate between the active sites containing thiamine pyrophosphate (TPP) and pyruvate (Pyr) on Elt CoA on E2, and FAD on E3. Acetyl-CoA and GTP are allosteric effectors of E, and NAD+ is an inhibitor of the overall reaction.

MECHANISM FIGURE 16-16 The role of biotin in the reaction catalyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the e-amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed by separate active sites on the enzyme as exemplified by the pyruvate carboxylase reaction. In the first phase (steps to ), bicarbonate is converted to the more activated C02, and then used to carboxylate biotin. The bicarbonate is first activated by reaction with ATP to form carboxyphosphate (step ), which breaks down to carbon dioxide (step ). In effect, the... 

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