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Isozymes Provide a Means of __Specific to __ and __
· Isozymes are __; they display different __, or __. They are encoded by __, which usually arise through __ and __
- Regulation
- Distinct Tissues and Developmental Stages
- enzymes that differ in amino acid sequence yet catalyze the same reaction
- kinetic parameters, like KM
- respond to different regulatory molecules
- different genes
- gene duplication and divergence
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· The existence of isozymes permits the __ to meet the needs of a given tissue or developmental stage
o Ex: lactate dehydrogenase (LDH) catalyzes a step in __ and __. Humans have __for this enzyme: the __ and the __. They are __ identical. Each functional enzyme is __, and many different combos of the two __polypeptide chains are possible.
- fine-tuning of metabolism
- anaerobic glucose metabolism and glucose synthesis
- two isozymic polypeptide chains
- H isozyme is highly expressed in heart muscle and the M isozyme is expressed in skeletal muscle
- 75%
- tetrameric
- isozymic
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§ The __, found in the heart, has a __ than does the __. The two isozymes also differ in that __. The other combos, like H3M, have intermediate properties.
- H4 isozyme
- higher affinity for substrates
- M4 isozyme
high levels of pyruvate allosterically inhibit the H4 but not the M4 isozyme
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· A donor molecule provides the __being attached. Most modifications are __, such as __ and __.
o The __ and __ enzymes are themselves regulated by __, showing that the __ of a protein can be controlled by the __ of the modifying enzymes
- functional moiety
- reversible
- phosphorylation and dephosphorylation and acetylation and deacetylation
- acetyltransferase and deacetylase
- phosphorylation
- covalent modification
- covalent modification
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· Modification isn’t always __, such as adding __ for __, such as __(a GTPase) and __(a protein tyrosine kinase), to become __, allowing them to better __ and __ info that is being passed along their __.
- reversible
- lipid groups for signal-transduction pathways
- Ras
- Src
- affixed to the cytoplasmic face of the plasma membrane
- receive and transmit info
- signaling pathways
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o Attachment of the small protein __can signal that a protein is to be destroyed, the ultimate means of regulation. The protein __must be __and destroyed before a cell can enter __and proceed through the cell cycle
- ubiquitin
- cyclin
- ubiquinated
- anaphase
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· __ and __ control the extent of __
o As much as 30% of eukaryotic proteins are __ by __, one of the largest families; it allows regulation to be what
- Kinases and phosphatases
- protein phosphorylation
- phosphorylated by protein kinases
- fine-tuned according to a specific tissue, time, or substrate
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· ATP is the most common donor of __. The terminal group of ATP is transferred to a __, which is commonly __, __, or __. Transfers to __ and __ residues are handled by one class of protein kinases and to __residues by another.
o Tyrosine kinases play pivotal roles in growth regulation, and mutations in these enzymes are commonly observed in cancer cells
- phosphoryl groups
- specific amino acid
- commonly serine, threonine, and tyrosine
- serine and threonine
- tyrosine
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The acceptors in __ are located inside cells, where the __is abundant. Proteins that are entirely extracellular are not regulated by __.
- protein-phosphorylation reactions
- phosphoryl-group donor ATP
- reversible phosphorylation
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· Protein kinases vary in their degree of __
o Dedicated protein kinases phosphorylate a __ or __
o __ modify many targets; they have a wide reach and can coordinate diverse processes
§ A __ recognizes related sequences. For example, the __ recognized by __ is __, where X is a __ and Z is a __, and __ and __ are the sites of __
· This sequence is not required. Lysine can sub for one of the Arg residues, with a decrease in affinity
- specificity
- single protein or several closely related ones
- Multifunctional protein kinases
- multifunctional kinase
- consensus sequence
- protein kinase A (PKA)
- Art-Arg-X-Ser/Thr-Z
- small residue
- large hydrophobic one
- Ser and Thr
- phosphorylation
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§ Short synthetic peptides containing a __ are nearly always __by __. Thus, the __ is the __ surrounding the __.
· Distant residues can, however, contribute to __.
- consensus motif
- phosphorylated
- serine-threonine protein kinases
- primary determinant of the specificity
- amino acid sequence
- serine or threonine phosphorylation site
- specificity
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· Protein phosphatases do what and how? The enzyme does what?
o The unmodified __is regenerated and __is produced. These enzymes play a vital role in cells because they __ that are activated by __.
- reverse the effects of kinases by catalyzing the removal of phosphoryl groups attached to proteins
- hydrolyzes the bond attaching the phosphoryl group
- hydroxyl-containing side chain
- orthophosphate
- turn off signaling pathways
- kinases
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· __ and __ are not the reverse of each other; both are irreversible and take place at negligible rates without the enzyme
o So, __will take place only through the action of a specific __ and at the expense of __, and __will take place only through the action of a __
§ Result: target proteins cycle __between __ and __ forms; the __ depends on the activities of __ and __
- Phosphorylation and dephosphorylation
- phosphorylation
- protein kinase
- ATP cleavage
- dephosphorylation
- phosphatase
- unidirectionally
- unphosphorylated and phosphorylated
- rate of cycling
- kinases and phosphatases
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· Reasons why phosphorylation is a highly effective means of controlling the activity of proteins
- o The free energy is large; and, it can change the conformational equilibrium between different functional states
- o A phosphoryl group adds two negative charges to a modified protein, disrupting electrostatic interactions and forming new ones
- o A phosphoryl group can form three+ hydrogen bods. These bonds are highly directional
- o Phosphorylation and dephosphorylation can take place in less than a second
- o Phosphorylation often evokes highly amplified effects
- o ATP is the cellular energy currency
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· __ activates __ by altering the __
- Cyclic AMP
- protein kinase A
- quaternary structure
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What is cAMP?
o cAMP is an intracellular messenger formed by cyclization of ATP; and, it activates a key enzyme, PKA, which alters the activities of target proteins by phosphorylating specific serine or threonine residues.
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§ Most effects of cAMP in eukaryotic cells are achieved through __
· __is activated by __concentrations near 10 nM; it has __.
o In the absence of cAMP, what happens?
activation by cAMP of PKA
- PKA
- cAMP
- two subunits—a 49 kD regulatory subunit and a 38 kd catalytic subunit
the regulatory and catalytic subunits form an R2C2 complex that is enzymatically inactive
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§ When two molecules of __bind to each of the regulatory subunits, the __ dissociates into an __ and __. These free catalytic subunits are then active.
· Thus, the binding of cAMP to the regulatory subunit does what?
o __and most other kinases exist in __ fo __ to meet the needs of a specific cell or developmental stage
- two
- cAMP
- R2C2
- R2 subunit and two C subunits
- relieves its inhibition of the catalytic subunit
- PKA
- isozymic forms for fine-tuning regulation
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· How does the binding of cAMP activate the kinase?
- o Each R chain contains the sequence Arg-Arg-Gly-Ala-Ile, which matches the consensus sequence for phosphorylation except for the presence of alanine in place of serine. In the R2C2 complex, the pseudosubstrate sequence of R occupies the catalytic site of C, thereby preventing the entry of protein substrates
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§ The binding of cAMP to the R chains does what?
§ The released __ are then free to __ and __.
- allosterically moves the pseudosubstrate sequences out of the catalytic sites
- C chains
- bind and phosphorylate substrate proteins
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· ATP and the target protein do what?
o The 350-residue catalytic subunit of __has two lobes. __ and __ fill a deep cleft between the lobes. The __lobe makes many contacts with __, whereas the larger lobe binds the peptide and contributes the key catalytic residues.
- bind to a deep left in the catalytic subunit of PKA
- PKA
- ATP and part of the inhibitor
- smaller
- ATP-Mg2+
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§ As with other kinases, the two lobes move closer to one another on __; mechanisms that restrict this domain closure provide the __
· The PKA structure has broad significance because residues 40 to 280 constitute a __that is common to all known protein kinases
- substrate binding
- regulation
- conserved catalytic core
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