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Determining Km and Vmax
(1/V)=(1/Vmax)+(Km/Vmax) (1/S) results in a straight line
All of the enzymes that exist have the Km they do because of what?
- they have evolved to funciton in their physological environments
- They evolved to function perfectly well with those Km values
3rd definition of Km
under certain conditions (like when substrate concentrations are low) the Km value approximates the affinity of the substrate for teh enzyme
lower Km= higher affinity; don't need a lot of substrate to bind
When we're turning over substrate and making product, what is that?
When the substrate of S is low, what is E?
What do we want?
we want Vmax to be high; Km we want to be high; so the ratio will be high
What does chymotrypsin have a preference for?
large hydrophobic side chains
What controlles enzyme rates?
diffusion; the reactions happen only as quickly as diffusion will allow; it is the limiting facto
Explain the example lactate dehydrogenase.
two subunits which bind in an ordered sequental way, which is when there is more than one substrate that binds to the enzyme
they have to bind in a particular order; more complicated than a single substarte
doesn't matter which order they bind; doesn't matter which order they leave
Which enzymes follow MM kinetics?
What can't they explain?
single substrate, non-alloseteric.
ordered, random sequential, double displacement, allosteric
enzyme substrates that will never be in the active site at the same time; one displaces the other
bind to enzyme via weak interacts and can let go
form covalent bonds and, once that is done, enzyme can no longer react
Reversible: what are the three?
competitive, uncompetitive, and noncompetitve
binds in active site and competes with substrate for the active site
looks like substrate but is not
- doesn't bind in the absence of substrate
- substrate binds and then the uncompetitive inhibitor binds to the site on the enzyme htat appears only after substrate binds
If substrate is not there, there is no binding site
When bound, binding site apears
all weak interactions and position of amino acid side chains doesn't react
binds in different location at any time--> causes allosteric change in enzyme; so active site no longer properly boinds substrate; can bind at any time
How to tell the difference? Competitive?
In competitive, the inhibitor does not affect Vmax; Km is affected. Since reversible, substrate can displace inhibitor in active site; adding more inhibitor lowers the rate of the reaction, but Vmax can still be attained
Competitve inhibitors change the Km for the rxn. Vmax does not change
Amount of substrate needed to get half of Vmax is different
we're removing substrate from the environmnet because the enzyme is bound by the inhibitor, causing the substrate to be locked into an inactive enzyme
- There is a reduction in vmax short of the uninhibited Vmax.
- If we have 100 enzymes, for example, and inhibitor inactivates by binding, it's almost as if they don't exist. So, Vmax goes down
Km changes as well because, as we add inhibitor, it is binding substrate as wel, causing less substrate to be present
two things can happen: E+S-->ES or E+I--> EI
substrate can also bind, creating an ESI complex
Inhibitor can let go
Same Km, but Vmax is affected. The number of enzymes put in are not all available when inhibited. Vmax corresponds only to the enzymes that can react
Substrate unaffeted. Vmax different
What is it possible to do?
design drugs that bind irreversibly via covalent bonds between inhibitor and enzyme
recycle enzyme! nothing else to do
What are the three irreversible inhibitors
- group specific reagents
- affnity labels
- suicide inhibtiors
Group specific reagents?
what matters is that side chain reacts/ exists in active site
it gets in, and a reaction causes a bond to form, inactivating the enzyme and making it irreversible
ex: DIPF (inhibitor that reacts with active serine residues)
differ from GSR because they work like the substate and mimic it to get into the active site
Once in, a rxn. occurs such that covalent bond is formed btwen inhibitor and enzyme (reaction is not specific)
Because it look slike the substrate, it ets into the active site--> covalent bond forms, inactivating enzyme
get into active site and begins to react similarly to substrate
reaction intermediate forms covalent bond--> enzyme inactive-> reaction is specific-- inhibtor prevents this in Parkinson patients.
All irreversible inhibitors result in what?
an irreversible binding to the enzyme and rendering it inactive
What is an example of a suicinde inhibitor?
Explaint he bacterial cell wall?
- has sugar groups held together by amino acids
- repeating units of NAM and NAG
- protects from osmotic stress
- Strung amino acids that are connected via pentaglycine bridges
enzyme responsible for making cross links, without which the cell wall is useless, not wrapped around
one alanine is cleaved off and the remaining alanine forms a temp cov bond with enzyme
bring in the second substrate, whcih takes the trapped free energy, transfer to form cross link between glycine and alanine
What does penicillin do?
fits into the active site and has highly reactive bond that is similar to original substrate
the bond is broken--> covalent linkage to serine residues like alanine but its not alanine. So, alinie isn't released and there's no room for the second substrate
Total inactivation of enzyme; no crosslinks are made; no protected cell wall to keep bacteria from suffering from osmotic stress--> lysis--> YOU'RE CURED