BIO 172 -- Exam 1 -- Lecture 3

Enzyme regulation

Environmental Factors (temperature, pH etc.) IRREVERSIBLE Inhibition-inhibitor binds to enzyme covalently... will never leave bc covalent bonding is very strong REVERSIBLE Inhibition-non-covalent bonding -Competitive inhibitor -Noncompetitive inhibitor Allosteric Enzymes -- Reversible Inhibition- non-covalent bonding and there are two classes (competitive and noncompetitive) .. both are reversible Allosteric Enzymes are examples of noncompetitive inhibitors

Allosteric regulation

Enzyme activity can be regulated by positive or negative feedback, causing activation or deactivation of the enzyme according to the needs of the cell. Such regulation is called ALLOSTERIC regulation. Allosteric regulation occurs when a regulatory molecule (an ACTIVATOR or an INHIBITOR) binds AWAY from the active site, causing a CONFORMATIONAL CHANGE in the active site of the enzyme, such that substrate will either fit into the active site (activation), or it won't (inhibition). Often times, metabolites in a particular pathway will be allosteric activators or inhibitors to either turn on or turn off synthesis of themselves. -- regulated by positive or negative feedback arrows represent different enzymes allosteric inhibition is NONCOMPETITIVE inhibition

COFACTORS

Enzymes are LIMITED in their catalytic flexibility by the chemistry provided by the R groups of amino acids. (ie. none of the R groups can absorb visible light or bind oxygen as a carrier.) Many chemical reactions CANNOT be catalyzed by proteins alone. COFACTORS are additional factors that can work with proteins to provide a broader range of chemistry at the active site, allowing enzymes to catalyze a BROADER range of reactions. -- Cofactors = SMALL, non protein molecules. Usually inorganic (metal) ions like Zn2+, Mg2+, Fe2+. --May be organic vitamin derivatives like NAD+, FAD+. --Many are attached at active site -- Many interact directly with the substrate to lower activation energy -- cofactors are not proteins and usually enzymes can not work without their co-factor — many are metal ions cofactors are attached to the enzyme and usually it is crucial to any reaction ex: ex: hemoglobin and sickle cell disease

Enzyme kinetics

Km is higher for enzymes with lower affinity for substrate (less efficient) >> need increased [S] substrate has a higher affinity for the red enzyme bc Km for red is smaller... Purple enzyme is less efficient at producing product than red enzyme.(look at graph)

Km

Km is the substrate concentration at which the reaction rate is HALF maximum (1/2 Vmax). If an enzyme has a low Km, it achieves maximum catalytic efficiency at low substrate concentrations. The smaller the value of Km, the MORE efficient is the catalyst and the better the substrate for the catalyst Km is not a reaction rate... it is a measurement of substrate concentration [S] -- amount of substrate it takes to get to 1/2 Vmax AFFINITY an enzyme has for a substrate low Km == very good enzyme for substrate == more efficient, quicker Km = a substrate Concentration that it takes to get 1/2 Vmax Enzymes have varying tendencies to bind their substrates (affinities) -- An enzyme's Km describes the substrate concentration at which HALF the enzyme's active sites are occupied by substrate. A high Km means A LOT of substrate must be present to saturate the enzyme, meaning the enzyme has LOW affinity for the substrate. On the other hand, a LOW Km means only a SMALL amount of substrate is needed to saturate the enzyme, indicating a HIGH affinity for substrate.

Would increasing enzyme concentration affect KM?

NO! ...beacuse.... Changing the number of enzyme molecules doesn't alter the enzyme's chemical characteristics so you'd expect them to bind substrate no better and no worse than usual. KM is NOT affected by enzyme concentration... unchanged -- Vmax is proportional to enzyme concentration. enzyme concentration does not affect Km ... Km should be unchanged 2 enzyme: 1 substrate 4 enzyme: 2 substrate -- double enzymes, the rate of the reaction should double

Competitive Inhibitors

REVERSIBLE -Bear a CLOSE structural and chemical similarity to substrate - Compete with substrate for active site - ADDING substrate can OVERCOME inhibition -- 1. Competitive Inhibitors — ex: white chocolate resees vs chocolate recess - enzyme CANNOT distinguish between the different substrates... thinks they're the same - ADDING substrate can overcome inhibition Effect DECREASES with increasing substrate concentration -- Competitive inhibition directly blocks the active site... When the regulatory molecule binds to the enzyme's active site, the substrate cannot bind... Compete with S for active site of enzyme Often resemble S Bind to enzyme with WEAKER bonds Reese's white chocolate peanut butter cups competitive inhibitor is so chemically similar to the substrate but there will NOT be as strong bonds (non-covalent bonds) -- Vmax=same KM=higher. At very high [S] Substrate "outcompetes" the inhibitor. Vmax is the same. -- inhibitor is slower, but will eventually reach Vmax slower than substrate at high substrate concentration you can have the same Vmax and inhibitor will not matter With inhibitors, Vmax will stay the same but Km will increase bc they are slower

Non-competitive inhibition is an example of allosteric inhibition.

allosteric regulation-- Is not generally affected by changes in substrate concentration because nothing is binding to the active site -- allosteric activation = the active site becomes available to the substrates when a regulatory molecule binds to a different site on the enzyme -- allosteric deactivation = the active site becomes unavailable to the substrates when a regulatory molecule binds to a different site on the enzyme

Vmax

enzyme is processing substrate to product as fast as it can (saturation) max potential, max velocity = Vmax = saturation of the enzyme Vmax is reached when enzyme is processing substrate to product as fast as it can (saturation)

when going from peanut butter cup to sucrose, it is an.....

exergonic reaction.... however, we can speed up reaction rate by adding a catalyst such as an enzyme.

Example of calculating Km

if Vmax = 82, then 1/2 Vmax is 41 reaction rate, and that corresponds to 0.1 of substrate Km = 0.1 substrate [S] -- KM value is always the Substrate concentration ... one number take half Vmax so 41 and then find what the substrate concentration is at 41 velocity.... so answer would be about 0.1 concentration -- Km= [substrate] at Vmax/2

Enzyme Kinetics Varies with Substrate Concentration... What do you do to increase rate of reaction?

increase enzyme concentration... double enzyme concentration = double reaction rate... it is proportional

Scenario: 1 enzyme, 2 substrates Enzyme Km with substrate A=.005nm Enzyme Km with substrate B=.007nm Which substrate does the enzyme catalyze most efficiently?

substrate A catalyzes most efficiently if Km is low, the chemistry between substrate and enzyme is better (better affinity) and takes less energy

what is true of enzymes?

they stabilize the transition state... because they create a lower transition state aka more stable

how to graph enzyme kinetics w substrate concentration

x-axis = Substrate Concentration [S] y-axis = Reaction rate (velocity) Reaction rate (Velocity) = PRODUCT PER UNIT TIME Vmax is line of saturation at top of graph

NONCOMPETITIVE inhibitors

(reversible) -No similarity to substrate - Bind at a location other than active site and change shape of active site - Adding substrate CANNOT overcome inhibition -- 2. Noncompetitive Inhibitors- do not bind to active site, alters the shape of active site... then the normal substrate has a harder time binding to the enzyme - Adding substrate cannot overcome inhibition... Very high [S] does not change the effect of the inhibitor -- - Inhibitor binds away from active site of enzyme but alters the conformation of the active site, distorting active site so catalysis is very slow or cannot happen at all - Do NOT resemble substrate - Vmax = lower - Km = do not need to know (***Because non-competitive inhibitors work in different ways, you are not responsible what happens to Km in the presence of a non competitive inhibitor.)

How can we increase Vmax?

1) Increase Enzyme Concentration When every enzyme has substrate attached to it, Vmax is reached. If we double the number of enzymes, this means we have twice as many substrate molecules bound to active sites, so the reaction rate would double. **Vmax is proportional to enzyme concentration.

2 Classes of reversible enzyme inhibitors

1) competitive inhibitors 2) noncompetitive inhibitors

more info

1. Regulatory molecules can be activators or inhibitors; they activate or deactivate the enzyme by changing its conformation 2. Allosteric regulators bind non covalently at site other than active site = regulatory or allosteric site 3. The regulators alter properties of active site, such that the active site is available to bind to substrate (allosteric activation) or unavailable (allosteric inhibition or deactivation) -- everything is NON-COVALENT bonding when allosteric inhibitor binds, it distorts the active site and overall will get a LOWER Vmax allosteric activation (positive feedback) makes the active site available to substrate