Enzyme Regulation
Inhibition
– Three Types:
(1) Competitive
– inhibit at the active site (classical, in which inhibitor looks like the substrate) or elsewhere (allosteric)
concentration of both substrate and inhibitor affect rate
same V
max
since substrate can compete out inhibitor
higher apparent K
m
since there is an alternative reaction – K
m
changes by a factor of (1 + [I]/K
I
)
Lineweaver-Burke:
steeper, same y-intercept
(2) Non-competitive
– inhibit somewhere other than active site – two mechanisms, both result in a dead enzyme
(1)
generic deactivator
– e.g., lead destroys cysteine reactivity
(2)
suicide inhibitor
– specifically targets one enzyme
does not preclude enzyme from binding to substrate, but prevents its activity
less total enzyme, so
K
m
same but reduced V
max
(recall that V
max
= k
2
[E
T
])
Linewaver-Burke:
steeper, same x-intercept
(3) Uncompetitive
– only when enzyme binds more than one substrate (most do)
inhibitory binding site present only when enzyme bound to first substrate
inhibitor binds to ES
1
complex, preventing interaction with second substrate
can be competitive with second substrate
both K
m
and V
max
reduced
, but the relationship between them is unchanged
K
m
appears lower because inhibitor shunts ES and less enzyme is necessary to bind
Lineweaver Burke:
parallel and higher
Regulation of Enzyme Activity
– In addition to product inhibition, enzyme activity may be altered in two ways:
(1) Allosteric Effects
– result in
cooperativity
(like hemoglobin) – effector binding determines affinity state (low or high)
result in sigmoidal not asymptotic curve
no K
m
– S
0.5
is analog (middle of sigmoidal curve, half maximum velocity when substrate at this concentration, like K
m
)
allosteric effectors can affect the curve differently:
K effectors
– different S
0.5
, same V
max
, so curve shifted to the side – analogous to competitive inhibition
e.g., isocitrate dehydrogenase – increasing [ADP] moves the curve to the left
M effectors
– same S
0.5
, different V
max
, so curve shifted up or down – analogous to non-competitive inhibition
sometimes, each subunit of a protein can be affected differently
e.g., phosphofructokinase – at high ATP levels, enzyme is only active if AMP levels are also high
second site on the molecule binds ATP to inhibit – binding AMP rescues enzyme function
(2) Alteration in Specific Activity
specific activity
= (number of moles of product formed) / (per unit time (usu. mM/min) times enzyme molecules)
measure of how rapidly the enzyme can react
zymogens (proenzymes)
– synthesis of biologically inactive form cleaved by proteolysis to form active enzyme
irreversabile, so expensive – must make new protein after old is inactivated
phosphorylation
– PO
3
from ATP to serine or threonine residues – 10-20 fold difference in activity
reversable – can phosphorylate (kinases) and dephosphorylate (phosphatases) again and again
uses much less ATP than zymogens
one phosphorylation pathway: cAMP-dependent protein kinase (PKA) – only phosphorylates when cAMP around
under starving conditions, cAMP is elevated, and PKA is more active
thus, PKA is used to control metabolic flux (gluconeogenesis vs. glycolysis, etc.)