Phosphorylases active sites

Phosphonomannans, fungal, 394,417 Phosphorus, in yeast mannan, 391 Phosphorylases active sites of, 346 amino acids of, 345 kinetics, 289,353 mechanism of action of, 356 properties of, 347... 

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

Figure 2.5. Diagramatic representation of the goal of rational drug design (a) illustrates the normal catalytic activity exhibited by purine nucleoside phosphorylase (PNP) (b) represents an effective inhibitor of PNP, which hts well into the active site thereby blocking its normal enzymatic activity...

Phosphorylation of glycogen phosphorylase is the initiator for the coupled conformational changes, which are coimmmicated over a large distance to the active site. Similar to the allosteric mechanism of phophofructokinase, the inter-subunit contact sinfaces play a decisive role for the communication between the phosphorylation site... 

The breakdown of glycogen in skeletal muscles and the liver is regulated by variations in the ratio of the two forms of glycogen phosphorylase. The a and b forms differ in their secondary, tertiary, and quaternary structures the active site undergoes changes in structure and, consequently, changes in catalytic activity as the two forms are interconverted. 

Feedback can also be positive. Since AMP is a product of the hydrolysis of ATP, its accumulation is a signal to activate key enzymes in metabolic pathways that generate ATP. For example, AMP causes allosteric activation of glycogen phosphorylase, which catalyzes the first step in the catabolism of glycogen. As is shown in Fig. 11-5, the allosteric site for AMP or IMP binding is more than 3 nm away from the active site. Only a... 

Figure 12-5 (A) Stereoscopic view of the structure of the catalytic site of phosphorylase b in the inhibited T-state with the inhibitor nojirimycin tetra-zole bound into the active site. Inorganic phosphate (P ) as well as the coenzyme pyridoxal 5 -phosphate (PLP) are also shown. (B) Details of interactions of the inhibitor, P , and PLP with the protein and with water molecules (small circles). This is a weak-binding state but the P has displaced the negatively charged side chain carboxylate of Asp 283 (visible at the lower right in A).

The active sites of these enzymes can have a nitrogen ligand, usually as histidine (acid phosphatases and some protein phosphatases), a nucleophilic serine residue (alkaline phosphatases), a cysteine residue in which the thiol group can form a covalent species with the phosphate ester (protein phosphatases), or an aspartate-linked phosphate (plasma membrane ion pumps). The inhibitory form of vanadium is usually anionic vanadate V(V), but cationic vanadyl V(IV) has also shown strong inhibition of some types of phosphorylase reactions. Above neutral pH, speciation of vanadyl ions produces anionic V(IV) species capable of inhibition of enzymes in the traditional transition-state analogue manner [5],... 

A. The DADMe modification also provides a favorable charge localization to place the cation at the l -position. The pKa of l -pyrrolidine nitrogen in DADMe system is 8.575 and provides the cationic nitrogen to ion pair with phosphate in the active site of phosphorylases. The N4 nitrogen of Immucillin-H has a pKa of 6.972 and is cationic in the active site, however the Cl -C9 distance is only... 

Studies on the reactivation of apoglycogen phosphorylase with a variety of analogs of pyridoxal phosphate have shown that the catalytic moiety is the 5 -phosphate group - only analogs with a reversibly protonatable dianion in this position have any activity In the nonactivated form of phosphorylase b, the phosphate is monoprotonated (-OPO3H ) when the enzyme has been activated, either allosterically or by phosphorylation (phosphorylase a), it is dianionic (-OPOa ). A glutamate residue in the active site acts as the proton acceptor or donor for this transition between the inactive and active forms of the cofactor. 

Little is known of the active site of phosphorylases. As already mentioned earlier, pyridoxal 5-phosphate is essential for activity, but it is not believed to take part directly in the enzyme—substrate interaction. Indeed, a reduced form of the pyridoxal 5-phosphate—enzyme complex may be prepared without loss of phosphorylytic activity. ... 

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