Subunit structure catalytic sites

This model is based on subunit stoichiometry, predicted secondary structure and subunit-subunit interactions. Fi sector (a, /3, y, S, and e subunits) has catalytic sites and Fo sector (a, b and c subunits) forms a proton pathway. 

Figure 16.21 Structure of one subunit of the core protein of Slndbls virus. The protein has a similar fold to chymotrypsin and other serine proteases, comprising two Greek key motifs separated by an active site cleft. The C-terminus of the protein is bound in the catalytic site, making the coat protein inactive (Adapted from S. Lee et al., Structure 4 531-541, 1996.)...

The Na/K ATPase has been extensively purified and characterized, and consists of a catalytic a subunit of around 95 kDa and a glycoprotein 0 subunit of approximately 45 kDa (Skou, 1992). The functional transporter exists as a dimer with each monomer consisting of an a and /3 subunit. Hiatt aal. (1984) have su ested that the non-catalytic jS subunit may be involved in the cottect insertion of the a subunit into the lipid bilayer and, therefore, it is conceivable that a modification of the 0 subunit structure may be reflected by changes in the catalytic activity of the a subunit. Therefore, in studies involving the manipulation of tissue glutathione levels, alterations of intracellular redox state may have an effect on substrate binding at an extracellular site on this ion-translocating protein. 

The sodium and calcium pumps can be isolated to near purity and still exhibit most of the biochemical properties of the native pump. Some kinetic properties of these pumps in native membranes are altered or disappear as membrane preparations are purified. For example, when measured in intact membranes, the time-dependencies of phosphorylation and dephosphorylation of the pump catalytic sites exhibit biphasic fast to slow rate transition this characteristic progressively disappears as the membranes are treated with mild detergents. One suggested explanation is that, as the pumps begin to cycle, the catalytic subunits associate into higher oligomers that may permit more efficient transfer of the energy from ATP into the ion transport process [29, 30], Some structural evidence indicates that Na,K pumps exist in cell membranes as multimers of (a 3)2 [31]. 

In addition to the binding of substrate (or in some cases co-substrates) at the active site, many enzymes have the capacity to bind regulatory molecules at sites which are usually spatially far removed from the catalytic site. In fact, allosteric enzymes are invariably multimeric (i.e. have a quaternary structure) and the allosteric (regulatory) sites are on different subunits of the protein to the active site. In all cases, the binding of the regulatory molecules is non covalent and is described in kinetic terms as noncompetitive inhibition. 

The fact that ATP and CTP bind to the same site follows from the observation that adding ATP to the inhibited enzyme by CTP reduces or reverses the inhibition, presumably because ATP competes with CTP for the same site. The fact that CTP binds to an allosteric site (i.e., it is not a competitive inhibitor) follows from the so-called desensitization effect. Addition of mercurials [e.g., p-mercuribenzoate (PMB)] reduces or eliminates the inhibition by CTP. However, it has no effect on the enzymatic activity of ATCase, presumably because the mercurials affect the regulatory subunits but not the catalytic site. As for the mechanism of cooperativity (both positive and negative), it is known that CTP does induce changes in the quaternary structure of the enzyme. 

The structure of P pantotrophus (fomerly Thiospharea pantotropha, (3)) cytochrome cdi, the first of this type to have its crystal structure solved (4), shows that the enz5une is a homodimer of 567 aminoacid residues and each subunit contains both a c-type cytochrome center and a di heme center (Fig. 3). The di heme (Fig. 1) is unique to this class of enzyme and on that basis alone might be expected to be the catalytic site. The c-type cytochrome centers, which are defined by the covalent attachment of the heme to the polypeptide are usually, but... 

The concept of rotational catalysis by ATP synthase is based on (a) P and 0 exchange rate data attesting to strong cooperativity with sequential participation of several catalytic sites (b) Pi and ATP 0-isotopomer distributions indicating that all catalytic sites exhibit identical catalysis and (c) that catalysis is strongly influenced by the y-subunit whose primary structure was not likely to account for spatially similar interactions with the /3-subunits . The model was found to be compatible with the 2.8 A resolution structure of bovine heart mitochondrial Fi-ATPase. ... 

The structure and mechanism of catalysis of FTase were well defined in the late 1990s from several X-ray crystallography and elegant biochemical studies [24,26-30]. The enzyme is a heterodimer of a and P subunits [31,32]. The P subunit contains binding sites for both the farnesyl pyrophosphate and the CAAX protein substrates. A catalytic zinc (Zn " ) identified in the active site of the P subunit participates in the binding and activation of the CAAX protein substrates [28]. The Zn " is coordinated to the enzyme in a distorted tetrahedral geometry and surrounded by hydrophobic pockets [24,27]. Upon binding of the CAAX peptide, the thiol of the cysteine displaces water and is activated for a nucleophilic attack via thiolate on the C-1 carbon atom of farnesyl pyrophosphate [30]. 

In 1998, Pedersen, Amzel, and colleagues solved the X-ray structure of rat liver mitochondrial Fj-ATPase to 2.8 A resolution and obtained a more symmetrical structure of the a and (3 subunits [27]. In this structure, catalytic as well as non-catalytic sites are occupied with bound nucleotide and the three a subunits and the three (3 subunits are in very similar but distinct closed conformations, with no indication of an open conformation as found in the Walker structure. The rat liver crystals were grown in the presence of substantially higher concentrations of nucleotides, and the crystallization medium contained only ATP (and not AMP-PNP),butno Mg +. 

Alcohol dehydrogenases (ADH EC 1.1.1.1), for which several X-ray structures are available ", catalyze the biological oxidation of primary and secondary alcohols via the formal transfer of a hydride anion to the oxidized form of nicotinamide adenine dinucleotide (NAD ), coupled with the release of a proton. Liver alcohol dehydrogenase (LADH) consists of two similar subunits, each of which contains two zinc sites, but only one site within each subunit is catalytically active. The catalytic zinc is coordinated in a distorted tetrahedral manner to a histidine residue, two cysteine residues and a water molecule. The remaining zinc is coordinated tetrahedrally to four cysteine residues and plays only a structural role . 

Metabolic activators and inhibitors are structurally dissimilar to substrates. These effectors exert regulatory control over catalysis by binding at an allosteric site quite distinct from the catalytic site. Such heterotropic interactions are mediated through conformational changes, often involving subunit interactions. Allosteric effectors can alter the catalytic rate by changing the apparent substrate affinity (K system) or by altering the... 

Fig. 7. A pictorial representation of two views of the structure of aspartate transcarbamyl-ase. The hatched portions are regulatory subunits and the white portions are catalytic subunits. The binding sites for CTP ( ), carbamyl phosphate (O), and aspartate ( ) are indicated along with an SH group that is near the active site and a Zn atom that is required for the binding between regulatory and catalytic subunits.

FIGURE 20-32 A plausible model for the structure of cellulose synthase. The enzyme complex includes a catalytic subunit with eight transmembrane segments and several other subunits that are presumed to act in threading cellulose chains through the catalytic site and out of the cell, and in the crystallization of 36 cellulose strands into the paracrystalline microfibrils shown in Figure 20-29. 

Figure 9.9 shows the crystal structure of two of the subunits of phosphofructokinase from B. stearothermophilus. In the complete enzyme, the subunits are disposed symmetrically about three mutually perpendicular axes. Each of these axes is a twofold symmetry axis, which means that rotating the entire structure by half of a full circle (180°) around the symmetry axis results in an identical structure. This rotation is shown diagramatically in figure 9.10. Because ADP is a product of the enzymatic reaction as well as an allosteric activator, it binds at both the catalytic and allosteric sites (see figs. 9.9 and 9.10). The catalytic site for fructose-6-phosphate in each subunit is at the interface of the subunit with one of its neighbors, and the allosteric site is at the interface with a different neighbor. 

Interface between subunits A and D of phosphofructokinase near the catalytic site in (a) the T and (b) the R structures. Crystals of the enzyme in the R state were obtained in the presence of fructose-6-phosphate and ADP (see fig. 9.9) crystals in the T state were obtained in the presence of a nonphysiological allosteric inhibitor, 2-phosphoglycolate. The wavy green line represents part of the boundary between subunits A and D. The heavy green line indicates the polypeptide backbone. The side chains of Glu 161 and Arg 162 are shown in red. Note the inversion of the positions of these side chains in the two structures. (Source From T. Schirmer and P. R. Evans, Structural basis of the allosteric behaviour of phosphofructokinase, Nature 343 140, 1990.)... 

Subunit structure of aspartate carbamoyl transferase and the fragments produced by treating the enzyme with mercurials. In the complete enzyme (top), the three sets of regulator dimers are sandwiched between two trimers of catalytic subunits (see fig. 9.17). The approximate location of the active site in each c subunit of the trimer facing the viewer is indicated with a c. 

The structural transition to the T state disrupts the active site in two major ways. First, the domain of the c subunit that includes Arg 105 and His 134, which interact with carbamoyl phosphate, is pulled away from the domain that interacts with aspartate, because some of the residues in both domains are tied up in an alternative set of hydrogen bonds. Arginine 105 is hydrogen-bonded to Glu 50 in the same domain, instead of to the substrate His 134 interacts with a residue in the other c trimer. In addition, the loop of the c subunit that contains Ser 80 and Lys 84 is pulled out of the active site by hydrogen bonds to still another c subunit. A baroque net of interrelationships thus links the catalytic sites of all the c subunits in the complex. 

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