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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 __
- 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
The existence of isozymes permits the __ to meet the needs of a given tissue or developmental stage
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
The __, found in the heart, has a __ than does the __. The two isozymes also differ in that __. The other combos, like H3
M, have intermediate properties.
- H4 isozyme
- higher affinity for substrates
- M4 isozyme
high levels of pyruvate allosterically inhibit the H4 but not the M4 isozyme
A donor molecule provides the __being attached. Most modifications are __, such as __ and __.
The __ and __ enzymes are themselves regulated by __, showing that the __ of a protein can be controlled by the __ of the modifying enzymes
- functional moiety
- phosphorylation and dephosphorylation and acetylation and deacetylation
- acetyltransferase and deacetylase
- covalent modification
- covalent modification
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 __.
- lipid groups for signal-transduction pathways
- affixed to the cytoplasmic face of the plasma membrane
- receive and transmit info
- signaling pathways
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
__ and __ control the extent of __
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
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.
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
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
Protein kinases vary in their degree of __
Dedicated protein kinases phosphorylate a __ or __
__ 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
- single protein or several closely related ones
- Multifunctional protein kinases
- multifunctional kinase
- consensus sequence
- protein kinase A (PKA)
- small residue
- large hydrophobic one
- Ser and Thr
Short synthetic peptides containing a __ are nearly always __by __. Thus, the __ is the __ surrounding the __
Distant residues can, however, contribute to __.
- consensus motif
- serine-threonine protein kinases
- primary determinant of the specificity
- amino acid sequence
- serine or threonine phosphorylation site
Protein phosphatases do what and how?
The enzyme does what?
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
- turn off signaling pathways
__ and __ are not the reverse of each other; both are irreversible and take place at negligible rates without the enzyme
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
- protein kinase
- ATP cleavage
- unphosphorylated and phosphorylated
- rate of cycling
- kinases and phosphatases
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
__ activates __ by altering the __
- Cyclic AMP
- protein kinase A
- quaternary structure
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.
Most effects of cAMP in eukaryotic cells are achieved through __
__is activated by __concentrations near 10 nM; it has __.
In the absence of cAMP, what happens?
activation by cAMP of PKA
- 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
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?
__and most other kinases exist in __ fo __ to meet the needs of a specific cell or developmental stage
- R2 subunit and two C subunits
- relieves its inhibition of the catalytic subunit
- isozymic forms for fine-tuning regulation
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
§ 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
ATP and the target protein do what?
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
- ATP and part of the inhibitor
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
- conserved catalytic core