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· Regulation of enzymes allows them to __. There are five enzymatic forms of control.
WHat are the five?
function at proper time and place and allows coordination of activity
- allosteric
- multiple forms of enzyme
- reversible covalent modification
- proteolytic activation
- controlling amount of enzyme present
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Allosteric
- o contain distinct regulatory sites and multiple functional sites. Small signal molecules bind, allowing cooperativity to transduce information.
- § Ex: ATCase
- · Catalysis by aspartate transcarbamoylase of the first step is inhibited by CTP through feedback inhibition
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Multiple forms of the enzyme
o isozymes allow varying regulation of the same reaction at distinct locations or times to meet certain physiological needs in that particular tissue. They are homologous enzymes within a single organism that catalyze the same reaction but differ slightly in structure and KM and Vmax values
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Reversible Covalent Modification
o involve covalent attachment of a modifying group, usually phosphoryl groups from ATP, which is catalyzed by protein kinases and hydrolyzed by protein phosphatases
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proteolytic activation
- o the enzyme can be active or inactive; a different strategy is used to irreversibly convert an inactive enzyme into an active one
- § Many enzymes are activated by hydrolysis of a few peptide bonds or one in zymogens (proenzymes)
- · Ex: digestive enzymes such as chymotrypsin
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Controlling the amount of enzyme present
this takes place at the level of transcription
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· _ catalyzes the first step in the biosynthesis of pyrimidines: the __ to form __. This is the __ in the pathway that will ultimately yield pyrimidine nucleotides like CTP
- Aspartate transcarbamoylase
- condensation of aspartate and carbamoyl phosphate
- N-carbamoylaspartate and orthophosphate
- committed step reaction
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· Regulation occurs because __.
o The rate of the reaction catalyzed by __is fast at __but slows as __. Thus, the pathway continues to do what until what?
o This is __, inhibition of an enzyme by the end product of the pathway; it ensures by __ and intermediates are not formed when pyrimidines are abundant
- CTP inhibits ATCase
- ATCase
- low concentrations of CTP
- CTP concentration increases
- make new pyrimidines
- enough CTP is present
- feedback inhibition
- N-carbamoylaspartate
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· CTP is structurally different from the substrates of the reaction and must bind to site different from active site, called an __. It is an __
· ATCase consists of __ and can be separated by treatment with a __compound, which reacts with __.
o __after reveals the __, which can be separated by __ due to different charge or by __due to different size
- allosteric or regulatory site
- allosteric inhibitor
- separable catalytic and regulatory subunits
- mercurial
- sulfhydryl groups
- Ultracentrifugation
- two subunits
- ion-exchange chromatography
- centrifugation
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· The larger subunit is the __, which displays __activity but is __ and does not display __
o Consists of __; there are __
· The isolated smaller subunit can bind __, but has no __ activity, which is why it __
Consists of __; there are __
- catalytic subunit
- catalytic
- unresponsive to CTP
- sigmoidal kinetics
- three chains
- two catalytic trimers
- CTP
- catalytic
- regulates
- two chains
- three regulatory dimers
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· ATCase is composed of discrete __ and __, and the interaction of the subunits in the native enzyme produces its __ and __
Allosteric interactions in ATCase are mediated by __
- catalytic and regulatory subunits
- regulatory and catalytic properties
- large changes in quaternary structure
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o Two catalytic trimers are arranged how? There are significant contacts between the __ and __ subunits: each __ within a __ interacts with a __ within a __
The __ makes contact with a structural domain in the __ that is stabilized by a __ion bound to __ residues
- stacked one on top of the other, linked by three dimers of the regulatory chains
- catalytic and the regulatory subunits
- r chain
- regulatory dimer
- c chain
- catalytic trimer
- c chain
- r chain
- zinc
- four cysteine
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· To locate the active sites, the enzyme was crystallized in the presence of __, a bisubstrate analog that resembles an intermediate along the pathway of catalysis
o __is a potent __ of ATCase. It binds at sites where, each of which contributes __ to the complete enzyme
- PALA
- PALA
- competitive inhibitor
- lying at the boundaries between pairs of c chains within a catalytic trimer
- three active sites
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o Further examination of the ATCase-PALA complex reveals a remarkable change in quaternary structure on binding of PALA: What are these three changes?
§ the two catalytic trimers move 12 angstroms farther apart and rotate approximately 10 degrees about their common threefold axis of symmetry
§ also, the regulatory dimers rotate approximately 15 degrees to accommodate the motion
§ the enzyme expands
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o In essence, ATCase has two distinct quaternary forms: one that __ and another that __
- predominates in absence of substrate or analogs
- predominates when substrates or analogs are bound (T and R, respectively)
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· The enzyme exists in __between the T and R state. In absence of substrate, almost all enzyme molecules are in __, with a __ and __.
o Binding of substrate to one active site has two effects aside from increasing likelihood that entire enzyme shifts to R state: What is the first?
- equilibrium
- T state
- low substrate affinity and low catalytic activity
§ Increases probability that each enzyme molecule will bind at least one substrate molecule
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What is the second?
- § Increases average number of substrate molecules bound to each enzyme; presence of additional substrate will increase the fraction of enzyme molecules in the more active R state because the position of the equilibrium depends on the number of active sites that are occupied by substrate
- · The effects of substrates on allosteric enzymes= homotropic effects
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§ Basically, the enzyme is represented by a __mechanism
· The __ for ATCase is characteristic of a mix between the T and R state curves
o Increasing substrate concentration favors a __; and, when switching from T to R, the curve depends on the __. It switches from T to R within a __
- concerted
- sigmoidal curve
- transition from the T-state curve to the R-state curve
- substrate concentration
- narrow range of substrate
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· In the T state, the __ hold the two __ sufficiently close to each other that __ and __ necessary for __ and __
· Allosteric regulators modulate the T to R transition
o The enzyme is in the T state when bound to CTP; a binding site for this nucleotide exists in each regulatory chain in a domain that does not interact with the catalytic subunit
§ Each active site is more than 50 angstroms from the nearest CTP-binding site
- regulatory dimers
- catalytic trimers
- key loops on their surfaces collide
- interfere with conformational adjustments
- high-affinity substrate binding and catalysis
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· Mechanism of CTP inhibition: __
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the binding of the inhibitor CTP shifts the equilibrium toward the T state, decreasing net enzyme activity and making it more difficult for substrate binding to convert the enzyme into the R state. More substrate will be required
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· ATP is an allosteric effector in that it __ because it __
o The effects of nonsubstrate molecules on allosteric enzymes (like CTP and ATP) are __effects
§ Substrates generate the __ (__effects), whereas regulators shift the__ (__ effects)
- increases the reaction rate
- competes with CTP for binding to regulatory sites, preventing inhibition of the enzyme
- heterotropic
- sigmoidal curve
- homotropic
- KM
- heterotropic
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o Explanations for why ATP increases ATCase activity
- § High ATP concentration signals high concentration of purine nucleotides in the cellà lead to balance of purine and pyrimidine pools
- § High ATP concentration indicates that energy is available for mRNA synthesis and DNA replication and leads to the synthesis of pyrimidines needed for these processes
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· The effects of __ and __ can be modeled by changing the value of L. For the __, the value of L, which equals T/R, increases from 200 to 1250. Thus, more substrate is required to shift to R. For the ATP-saturated form, L decreases to 70
- CTP and ATP
- CTP-saturated form
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