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Covalent modification with examples Flipbook PDF
Covalent modification with examples
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COVALENT ENZYME REGULATION Both reversible and irreversible covalent modification of enzymes plays important roles in regulation of enzyme function
1. Reversible covalent modification The modulation of enzyme activity by the attachment or release of small groups plays a very important role in metabolic control. Probably the most universal, and certainly the best understood, is the phosphorylation of specific serine, threonine or tyrosine groups 2. Irreversible covalent modification Proteolytic cleavage of specific peptide bonds is often used to activate enzymes. Since proteolysis is essentially irreversible, turning the activity off requires another mechanism, often binding of inhibitory proteins. 1) Reversible covalent modifications. Reversible covalent modifications require expenditure of energy and are often used in signaling from extracellular messages. In contrast, noncovalent interactions are reversible with no metabolic energy expended and sense conditions within a cell. Various types of covalent modifications are known, such as adenylylation, methylation, or attachment of lipids. Reversible covalent modifications that are known to alter enzyme activity include: a) Phosphorylation of serine, threonine or tyrosine and less frequently aspartate and histidine residues. Protein Phosphorylation: Enzymes catalyzing the transfer of a phosphate from ATP to a protein are known as kinases and those
catalyzing the hydrolytic removal of the phosphate group are known as phosphatases. Coordinated Regulation of Glycogen Synthesis and Breakdown • The mobilization of stored glycogen is brought about by glycogen phosphorylase, which degrades glycogen to glucose 1-phosphate • Glycogen phosphorylase provides an especially instructive case of enzyme regulation • It was one of the first known examples of an allosterically regulated enzyme and the first enzyme shown to be controlled by reversible phosphorylation • It was also one of the first allosteric enzymes for which the detailed three-dimensional structures of the active and inactive forms were revealed by x-ray crystallographic studies
Glycogen phosphorylase action:
Allosteric and Hormonal Regulation of Glycogen Phosphorylase • In the late 1930s, Carl and Gerty Cori discovered that the glycogen phosphorylase of skeletal muscle exists in two interconvertible forms: glycogen phosphorylase a, which is catalytically active, and initiates an enzyme cascade, in which a catalyst activates a catalyst, which activates a catalyst. • Such cascades allow for large amplification of the initial signal.
JL Jain Fundamentals of Biochemistry
• The rise in [cAMP] activates AMP dependent protein kinase, also called protein kinase A (PKA). • PKA then phosphorylates and activates phosphorylase b kinase……………..catalyzes the phosphorylation of Ser residues in each of the two identical subunits of glycogen phosphorylase, ……………activating it ……………… stimulating glycogen breakdown
• In muscle, o this provides fuel for glycolysis to sustain muscle contraction for the fight-or-flight response signaled by epinephrine o In liver, glycogen breakdown counters the low blood glucose signaled by glucagon, releasing glucose o These different roles are reflected in subtle differences in the regulatory mechanisms in muscle and liver. o The glycogen phosphorylases of liver and muscle are isozymes, encoded by different genes and differing in their regulatory properties.
Nelson and Cox – Lehninger’s principles of Biochemistry
Regulation of glycogen degradation (glycogenolysis) U Satyanarayana- Biochemistry
Nelson and Cox – Lehninger’s principles of Biochemistry
• In muscle, superimposed on the regulation of phosphorylase by covalent modification are two allosteric control mechanisms. • Ca2, the signal for muscle contraction, binds to and activates phosphorylase b kinase, promoting conversion of phosphorylase b to the active a form • Ca2 binds to phosphorylase b kinase through its subunit, which is calmodulin. • AMP, which accumulates in vigorously contracting muscle as a result of ATP breakdown, binds to and activates phosphorylase, speeding the release of glucose 1-phosphate from glycogen • When ATP levels are adequate, ATP blocks the allosteric site to which AMP binds, inactivating phosphorylase.
Acetylation of lysine or amino terminal groups • Acetylation of lysines in histones is important in regulation of gene expression • Addition of the acetyl group to a lysine removes its positive charge, weakening the binding of histones to the negatively charged DNA which, apparently, results in a conformation more favorable for transcription Methylation of glutamate or aspartate residues Nucleotidylation of tyrosine residues /adenylation The biosynthetic pathways to glutamate and glutamine are simple, and all or some of the steps occur in most organisms. The most important pathway for the assimilation of NH4+ into glutamate requires two reactions. First, glutamine synthetase catalyzes the reaction of glutamate and NH4+ to yield glutamine. This reaction takes place in two steps, with enzyme-bound glutamyl phosphate as an intermediate.
• Glutamine synthetase is found in all organisms. • In addition to its importance for NH4+ assimilation in bacteria, it has a central role in amino acid metabolism in mammals, converting toxic free NH4+ to glutamine for transport in the blood • In bacteria and plants, glutamate is produced from glutamine in a reaction catalyzed by glutamate synthase • α-Ketoglutarate, an intermediate of the citric acid cycle, undergoes reductive amination with glutamine as nitrogen donor • The activity of glutamine synthetase is regulated in virtually all organisms
• In enteric bacteria such as E. coli, the regulation is unusually complex. • The enzyme has 12 identical subunits of Mr 50,000 (Fig. 22–5) and is regulated both allosterically and by covalent modification • Alanine, glycine, and at least six end products of glutamine metabolism are allosteric inhibitors of the enzyme ADP ribosylation primarily of arginine residues.
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The biosynthetic pathways to glutamate and glutamine are simple, and all or some of the steps occur in most organisms.
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The most important pathway for the assimilation of NH4+ into glutamate requires two reactions.
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First, glutamine synthetase catalyzes the reaction of glutamate and ammonia to yield glutamine.
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This reaction takes place in two steps, with enzyme-bound ϒglutamyl phosphate as an intermediate
• Glutamine synthetase is found in all organisms. In addition to its importance for NH4+ assimilation in bacteria, it has a central role in amino acid metabolism in mammals, converting toxic free NH4+ to glutamine for transport in the blood • In bacteria and plants, glutamate is produced from glutamine in a reaction catalyzed by glutamate synthase. • Alpha-Ketoglutarate, an intermediate of the citric acid cycle, undergoes reductive amination with glutamine as nitrogen donor:
The net reaction of glutamine synthetase and glutamate Synthase
Nelson and Cox – Lehninger’s principles of Biochemistry
Nelson and Cox – Lehninger’s principles of Biochemistry
Nelson and Cox – Lehninger’s principles of Biochemistry
• Superimposed on the allosteric regulation is inhibition by adenylylation of (addition of AMP to) Tyr397, located near the enzyme’s active site • This covalent modification increases sensitivity to the allosteric inhibitors, and activity decreases as more subunits are adenylylated. • Both adenylylation and deadenylylation are promoted by adenylyl transferase (AT), part of a complex enzymatic cascade that responds to levels of glutamine, α-ketoglutarate, ATP, and Pi.
• The activity of adenylyl transferase is modulated by binding to a regulatory protein called PII, and the activity of PII, in turn, is regulated by covalent modification (uridylylation), again at a Tyr residue. The adenylyltransferase complex with uridylylated PII (PII-UMP) stimulates deadenylylation, whereas the same complex with deuridylylated PII stimulates adenylylation of glutamine synthetase. • Both uridylylation and deuridylylation of PII are brought about by a single enzyme, uridylyltransferase. Uridylylation is inhibited by binding of glutamine and Pi to uridylyltransferase and is stimulated by binding of alpha-ketoglutarate and ATP to PII • The regulation does not stop there. The uridylylated PII also mediates the activation of transcription of the gene encoding glutamine synthetase, thus increasing the cellular concentration of the enzyme; the deuridylylated PII brings about a decrease in transcription of the same gene. • This mechanism involves an interaction of PII with additional proteins involved in gene regulation. • The net result of this elaborate system of controls is a decrease in glutamine synthetase activity when glutamine levels are high, and an increase in activity when glutamine levels are low and alpha-ketoglutarate
and ATP (substrates for the synthetase reaction) are available. • The multiple layers of regulation permit a sensitive response in which glutamine synthesis is tailored to cellular needs. Reference Nelson and Cox – Lehninger’s principles of Biochemistry