BIOCHEMISTRY - L. Stryer - 1984

VOLUME 2

PART II GENERATION AND STORAGE OF METABOLIC ENERGY

CHAPTER 16. GLYCOGEN AND DISACCHARIDE METABOLISM

16.11. Phosphorylase is Activated by the Phosphorylation of a Specific Serine Residue

Skeletal Muscle phosphorylase exists in two interconvertible forms: active phosphorylase a and generally inactive phosphorylase b (Fig. 16.5). The enzyme is a dimer with a subunit molecular mass of 92 kDa. Phosphorylase b is converted into phosphorylase a by the phosphorylation of a single Serine residue (serine-14) in each subunit. This covalent modification is catalyzed by a specific enzyme, phosphorylase kinase, which was discovered by Edmond Fischer and Edwin Krebs. Phosphorylase a is inactivated by a specific phosphatase that hydrolyzes the phosphoryl group attached to serine-14.

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Fig. 16.5. Schematic diagram of the Regulation of Glycogen phosphorylase in skeletal muscle. The enzyme can adopt a catalytically inactive T (tense) conformation or an active R (relaxed) conformation. The R ⇄ T equilibrium for phosphorylase a is shifted far toward the active R state. In contrast, phosphorylase b exists predominantly in the inactive T state, except when AMP levels are high and ATP and glucose-6-phosphate levels are low. Under most physiological conditions, the fraction of active enzyme is determined by the rates of phosphorylation and dephosphorylation.

Muscle phosphorylase b is active only in the presence of high concentrations of AMP, which acts allosterically. AMP binds to the nucleotide-binding site and alters the conformation of phosphorylase b. ATP acts as a negative allosteric effector, competing with AMP. Glucose-6-phosphate also inhibits phosphorylase b, primarily by binding to a different Active Site. Under most physiological conditions, phosphorylase b is inactive due to the inhibitory effects of ATP and glucose-6-phosphate. In contrast, phosphorylase a is fully active regardless of the levels of AMP, ATP, and glucose-6-phosphate. The fraction of active enzyme is determined primarily by the rates of phosphorylation and dephosphorylation. In resting muscle, almost the entire

enzyme is in the inactive b form. As will be discussed in the next section, elevated epinephrine levels and electrical stimulation of the muscle lead to The formation of the active a form.

16.12. Three-Dimensional Structure of Glycogen Phosphorylase

X-ray crystallographic studies of the a and b forms of Glycogen phosphorylase have greatly facilitated the investigation of the catalytic and regulatory mechanisms of this key metabolic enzyme. The 841 amino acid residues of the monomeric subunit are compactly folded into three Structural domains (Fig. 16.6): an amino-terminal

domain (310 residues), a glycogen-binding domain (160 residues), and a carboxy-terminal domain (371 residues). The catalytic site is located in a deep cleft formed by amino acid residues from each of these three domains. Protecting the active site from the aqueous environment undoubtedly favors phosphorolysis over Hydrolysis. Pyridoxal phosphate (vitamin B₆), which is essential for enzyme activity, binds near the glucose-1-phosphate attachment site. The aldehyde group of this cofactor forms a Schiff base with the Lysine side chain of the C-terminal domain. The retention of enzymatic activity following the reduction of the Schiff base by borohydride indicates that, unlike in other pyridoxal Enzymes, the aldehyde group in this case does not participate directly in catalysis. At the same time, the phosphoryl group of pyridoxal phosphate appears to take a direct part in catalysis. The phosphorylase molecule also contains a glycogen-binding site located 30 Å away from the catalytic center. This site is crucial for the attachment of the enzyme to the glycogen particle. Because of this large distance between the glycogen-binding site and the catalytic center, the enzyme can carry out the phosphorolysis of many terminal residues without undergoing dissociation and reassociation after each catalytic cycle.

Fig. 16.6. Schematic representation of the α-carbon backbone of phosphorylase a. The localization of the catalytically active site, as well as the glycogen particle-binding sites, allosteric sites, and phosphorylation sites, is shown.

In addition, phosphorylase contains at least two allosteric control sites. Glucose and nucleosides, which are allosteric inhibitors of Liver phosphorylase a (Section 16.18), bind near the catalytic center. AMP, an allosteric activator of phosphorylase b, binds near the subunit interface, far from the catalytic center and the glycogen-binding site. Serine-14, the site of phosphorylation during The conversion of phosphorylase b to phosphorylase a, is also located near the subunit interface. This phosphoryl group in phosphorylase a is hydrogen-bonded to the side chain of Arginine-69. Meanwhile, the region encompassing the 19 N-terminal residues in phosphorylase b lacks a well-defined Structure. This region of the b form resembles in flexibility the highly mobile activation domain of trypsinogen, which adopts a strictly ordered conformation upon conversion into Trypsin (Section 8.11). Detailed studies of the structural changes induced by phosphorylation and allosteric effectors are currently ongoing. However, it is already clear that glycogen phosphorylase serves as a sophisticated integrator of information regarding cellular METABOLISM/26.html">Energy Metabolism.

16.13. Phosphorylase Kinase is Also Activated by Phosphorylation

The activity of phosphorylase kinase is also regulated by covalent modification. Like phosphorylase, phosphorylase kinase is converted from a low-activity form to a highly active form through phosphorylation. The enzyme that catalyzes this activation is a component of the hormone–cyclic AMP system, which we will discuss shortly. Phosphorylase kinase can also be partially activated via another pathway, by Ca²⁺ concentrations on the order of 10^-7 M. This activation mechanism is of vital biological importance because Muscle contraction is triggered by the release of Ca²⁺ (Chapter 34). Thus, glycogen breakdown and muscle contraction are linked by a transient increase in cytoplasmic Ca²⁺ levels.