The Behavior of Proteins: Enzymes, Mechanisms & Control – Part I
Chapter 7 - pgs. 171-183Outline
• the behavior of allosteric enzymes - concerted vs. sequential models • the regulation of enzymatic activity - phosphorylation and zymogens
• Michaelis-Menten kinetics is invaluable when dealing with simple, nonallosteric enzymes - KM, Vmax, kcat are hugely important values and characteristics to understand
• but, many enzymes are ALLOSTERIC - do we remember what that means…?
• Michaelis-Menten kinetics does not apply to these enzymes - the calculations, assumptions, predictions all fall apart
• but, we still must understand these more complicated allosteric enzymes if we are to understand something about biochemistry
• so, we will consider them today
Allosteric Enzymes and Cooperativity
• many allosteric proteins show ______________________________________ - not just enzymes
• binding of one molecule to a COMPLEX can – and often does – affect ___________________ structure
- side chains and other groups of the protein itself move to accommodate ligand/substrate binding
- these movements induce other movements and next thing you know the whole protein is changing its 3D shape
- a ____________________________________ CHANGE - someone fainting at the inauguration…
• _______________________ COOPERATIVITY is when ligand/substrate binding causes the protein to change its shape in a way that improves its activity or function
- ATCase: increase its enzymatic activity - hemoglobin: bind more O2 more easily
Regulation of Allosteric Enzymes
• aspartate transcarbamoylase (ATCase) catalyzes the first step (reaction) of a series of reactions that ultimately make CTP (cytidine triphosphate) – one of the 4 bases that make DNA and RNA • now – COMMON SENSE ALERT – making the building blocks of D/RNA are very expensive for
the cell (the cell’s currency is __________________) - ATP is the high energy molecule used for almost all things - ATP is also the A of D/RNA
- CTP, GTP, and TTP are all just as high energy as ATP and just as costly to make
- do you really want to be making more of them right now…? • the cell only wants to make CTP when it needs CTP
- makes sense…
• if you’re trying to determine if you need milk, do you check the butter? - do you check the bread…?
- you check the…
• the cell does the same thing!
- to determine whether or not it should make CTP, it checks the levels of CTP already present • it all makes sense!!!
• CTP itself directly ___________________ ATCase
- this is called __________________________ INHIBITION
- when the product of a series of reactions inhibits one or more of those reactions - you got it already? shut it down, save your energy for later!
• feedback inhibition is not a unique feature of allosteric enzymes • we’ll see it again and again when we discuss metabolism
• ATCase catalyzes the ‘___________________________’ of aspartate and carbamoyl phosphate
- the graphical representation of this reaction is ______________________ (curvy) - trademark of ALLOSTERIC enzymes
- do you remember what the graphical representation of nonallosteric enzymes looks like…? - ‘hyperbolic’
- the ATCase graph is sigmoidal due to _______________________ cooperativity
- velocity picks up as ________________________ is added • CTP inhibits the activity of ATCase dramatically
- the curve is still sigmoidal, because we have not affected cooperativity
- but, it takes much more substrate to get to the same velocity as the uninhibited reaction • with enough substrate, we can achieve Vmax
- sounds like a competitive inhibitor (from Monday’s lecture) - but, NO!!!!
- that was all based on Michaelis-Menten kinetics - and this is NOT Michaelis-Menten kinetics because….
… we’re dealing with an allosteric enzyme
• competitive inhibition also doesn’t make sense because CTP is a very different molecule from aspartate – it can’t fit in the active site • so, what’s going on…?
• ATCase is made up of two different types of subunits (six of each):
- two trimers of _____________________________ subunits
- three dimers of _______________________________ subunits
• if you separate these two types, the catalytic subunits still retain their activity - but the curve of the reaction is no longer sigmoidal (nonresponsive to CTP)
- in other words, without the regulatory subunits, ATCase loses its allosteric-ness - and becomes a Michaelis-Menten enzyme
- the regulatory subunits confer allosteric regulation • we’ve isolated the inhibition
- CTP must bind to the regulatory subunits to slow the enzyme • but, another interesting thing occurs
- in the presence of ATP, the reaction ______________________________ - the shape/structure of ATP and CTP are very similar
- but, ATP is not made from this pathway (only CTP, and TTP)
• however – COMMON SENSE ALERT – if we use ATP, GTP, CTP, and TTP primarily to make DNA and RNA, what do you think would be better: to have all four balanced or to have a lot more of some versus other…?
• it’s all about “________________________ the NTP pools” - if there’s enough ATP to bind to ATCase, then we need more CTP
• what’s the shape of the curve in the presence of the activator, ATP…? • when activated, the reaction is less COOPERATIVE
• COOPERATIVITY is a property of allosteric enzymes that allows the reaction to proceed more easily
- that implies there was some __________________ to the reaction causing it to naturally occur less easily (substrate binding…)
- under ACTIVATION, it’s pedal to the floor - all barriers lifted
- let this enzyme crank as fast as it can - we need CTP, and we need it NOW!
• no cooperativity – no sigmoidal curve – no allosteric-ness • CTP and ATP are very similar in structure
- they each compete for access to the ____________________________ binding site - DON’T CONFUSE THIS WITH COMPETITIVE INHIBITION!
• when CTP levels are high, it wins and slows the enzyme down - good, we got enough CTP – don’t make more
• when ATP levels are high, it wins and speeds the enzyme up - good, we got too much ATP – we need more CTP to balance it ATP inhibits the inhibitor!!
Some Key Terms
• it’s important to emphasize that what makes allosteric enzymes ‘allosteric’ is that they change their conformation (3D shape) when bound by different molecules
- literally translates to ‘other shape’
• these changes in shape lead to changes in function because… STRUCTURE=FUNCTION
• any molecule that binds an allosteric protein and induces a conformational change is called an
ALLOSTERIC ___________________________ - substrate, inhibitor, activator
• ____________________________ effects are when different molecules are involved - e.g., CTP and ATP binding ATCase – where aspartate is substrate
Concerted vs. Sequential Models for Allosteric Enzymes
• these are the two most widely used models for the behavior of allosteric enzymes (just as Michaelis-Menten is a model for non-)
• the CONCERTED MODEL states that an allosteric enzyme has two conformations:
- the _____________ form: R (relaxed) form – binds substrate tightly
- the ______________ form: T (tight, taut, tense) form – binds weakly
• the model also states that the conformations of all subunits switch from one form to the other
_______________________________________
- this is the concerted model’s distinguishing characteristic
• both subunits change from T (inactive) to R (active) at the same time
• there is an equilibrium between the R and T forms (as there is an equilibrium for most things that switch) with T favored
- but, affinity for substrate is ___________________ in the R form
• according to this model, all allosteric binding does is influence and change the _____________ between the T and R forms
• substrate searching for and finding open active sites results in it binding to the few R forms that are available
- this shifts the equilibrium and causes the R form to be favored - more R form, more substrate binding, more activity
• shifting equilibria…
• by effectively removing free R form from the equilibrium, we shift that equilibrium to the left and make more R form enzymes
• this shifting of the equilibrium gives the allosteric, sigmoidal effect
• ___________________ bind to and stabilize the T form; ________________ bind to the R form • the sequential model shares the conformational changes, R and T forms with the concerted
model
- however, the sequential model predicts that substrate finds a T form subunit and – at sufficiently high concentrations – can muscle its way into the active site anyway - shifting that active site from T to R
- this induces a conformational change in the complex that makes it _____________ for another substrate to bind to another site
- hence, cooperativity
• but T to R transitions are happening one at a time, not simultaneously
Summary
• allosteric interaction is when binding of one molecule to a complex affects ________________
• ___________________ COOPERATIVITY is when substrate binding causes the enzyme to change its shape in a way that improves its activity
• _______________________ INHIBITION is when the product of a series of reactions inhibits one or more of those reactions
- this often keeps energy from being wasted • ATCase as an example of an allosteric enzyme
• the CONCERTED MODEL states that an allosteric enzyme has two conformations – R and T –
and conformations of all subunits switch from one form to the other ______________________ • the SEQUENTIAL MODEL predicts that substrate finds a T form subunit and binds to it shifting
