Term 2 Lecture 4: Catalysis (Part 2) Flashcards
4 strategies used by enzymes in catalysis
All reduce Eact but do not change overall ∆G - just speed up the reaction
1) covalent catalysis - formation of chemical bonds between enzyme & substrate broke in formation of products covalent intermediates can be trapped but transition states cannot as they are unstable and exist fleetingly
2) catalysis by proximity - bringing substrates together to favour reaction
3) metal ion catalysis- uses metal ion cofactor in enzyme
4) general acid-base catalysis- proton donation and acceptable (aa side chains involved)
Reaction mechanism: the detailed sequence of steps in an enzyme catalysed reaction
Reaction mechanism: the detailed sequence of steps in an enzyme catalysed reaction
E.g.
Lactate dehydrogenase catalytic cycle
Pyruvate <->Lactate dehydrogenase<-> lactate (^ using cofactor NADH+H)
1) free enzyme E-H
2) NADH bound E-H-NADH
3) pyruvate bound E-H-NADH-pyruvate
4) redox reaction transition state
5) lactate bound E-NAD+ -Lactate
6) NAD+ bound E-H-NAD+
(NAD+ released returns to 1) state )
Region of enzyme that binds substrate(s) is called the active site
Active site is a crevice or cleft on the enzyme with a precise 3D shape determined by protein structure.
Generally a small part of the enzyme usually <10% of aa’s participate in forming the active site.
The rest of the proteins aas hold the active site together.
How substrates are bound to enzymes
by weak interactions (same as those in protein folding)
Interactions involve aa side chains and to a lesser extent the peptide backbone
Free energy of binding substrates range from ~ 15-50kj mol-¹ i.e. from energy of a strong H-H bond upto a weak non-cov bond
All types of weak interactions involved but most important are H-H and electrostatic interactions
Directional nature of H-H bonds and necessity of close proximity for various forces involving charges (inherent or induced) mean that complementary shape of active site and substrate is important for binding - leading to enzyme specificity
How enzymes decrease Eact
By decreasing the size of the “hump”
^by stabilising the transition state of the catalysed reaction
This can be achieved if the enzyme is able to form more favourable interactions with the transition state than with the substrate(s) or product(s)
Stabilisation of transition state may involve sig. decreases in energy - strong binding to the transition state can contribute to this but enzyme must not bind too strongly to substrate or product - if ES or EP too strong then they won’t come apart
Process:
E+S<->ES<->ES<->EP<->E+P
(Transitional state)
Shortened to:
E+S<>ES> E+P
The two scientists behind the Michaelis Mentin model
Maud Menten (1879-1960) was a biochemist who researched kinetics of enzyme catalysed reactions under supervision of Leonor Michaelis (1875-1949) and published findings in 1913. As a woman she could not conduct research in her home country of Canada at the time (Pre ww1) so moved to Europe (Berlin) and continued to publish papers until 1953.
Michaelis was a German biochemist, physical chemist and physician. His instructors at Berlin university included Emil Dubois Reymond (physiology,) Emil Fischer (chemistry) and Oscar Hertwig (histology/embryology). He attained a position at Berlin in 1903 became a professor in 1908 and in 1922 moved to university of Nagoya Japan as a professor of biochemistry - was one of the first foreign professors at a Japanese uni. 1926 moved to John Hopkins Uni in Baltimore, the. Rockefeller institute of medical research in NYC in 1929 where he retired in 1941
As well as his role in the Michaelis Menten model, Michaelis discovered thioglycolic acid could dissolve keratin - used in the cosmetic industry to perm hair
How was the Michaelis Mentin model discovered?
They used a partially purified preparation of “invertase” enzyme (EC32126) and added it to a solution of sucrose (table sugar)
Historical note: enzymes were aka as ferments as many were obtained from brewers or bakers yeasts
Sucrose is “optically active” and the enzyme reaction changes the “polarisation” of light passing through the sugar solution. This change in polarisation can be observed using a polarimeter (it has two polarisers each of which is like the lens in polarising sunglasses)
Sucrose+H2O >invertase> glucose+fructose
Rate of an enzyme catalysed reaction can be measured at different concentrations of reactant or substrate
Initial rate is estimated as the rate before [S] decreases due to build up of products inhibiting enzyme.
Rate of reactivity expressed as change in [S] per unit of time (usually s-¹) or change in amount of S per unit of time
Reaction velocity= rate of product formation=rate of substrate disappearance (stoichiometry)
Relationship between rate of reaction and substrate concentration
Is a hyperbolic curve for an enzyme catalysed reaction
Mathematically identical to that observed when binding of a ligand to a protein is analysed.
This relationship can only be explained if the curve results from binding of the reactant or substrate to the enzyme as part of the reaction
Kinetics of enzyme catalysed reactions differ from those of non-catalysed reactions
In absence of enzyme rate of reaction is proportional to [A] (ascending straight line)
In presence of enzyme, rate no longer shows linear dependence on [A] approaching a maximum value. Instead the rate now shows linear dependence on [E] the amount of enzyme present implying that enzyme-catalysed reaction involves formation of a complex between enzyme and reactant(s)
Explanation of Michaelis Mentin model
Kinetics of enzyme catalysed reactions can be analysed by this model.
It assumes that the formation of an enzyme-substrate complex is the first step in the reaction mechanism.
For the overall reaction
S (substrate) -> P (product)
Is catalysed by E (Enzyme)
Steps:
1) E+S<-> ES (formation of enzyme substrate complex - reversible.
2) ES→E+P (enzyme substrate complex to enzyme and product - assumed irreversible)
The second step is non-reversible
if [P] =~0
I.e. we can measure initial rate of reaction immediately after mixing E&S
Alternatively second step can be viewed as essentially irreversible under normal conditions used for enzyme analysis where [S]»[P]
Or where the reaction has large neg. free energy change in forward direction.
Steps can be combined and written
E+S <> ES > E+P
Transition state complex and state EP ignored here as (usually) short-lived
E+S > ES is kf forward constant forward reaction over 1st step
ES > E+S is reverse rate constant reverse reaction over 1st step
ES> E+P is kcat second step
Kcat is the limiting rate constant of any enzyme catalysed reaction at saturation
Vo = Vmax ( [S]/[S]+Km
By assuming that concentration of enzymes is much less than [S] and that formation of ES is faster than P - so that 1st step reaches equilibrium or ES reaches steady state we can determine that:
Vo = Vmax ( [S]/[S]+Km
Where:
Vo=initial rate of reaction(e.g. measured parameter
Vmax = Kcat[E] (a constant for an assay system) maximum rate
Km = (Kr+Kcat)/Kf (constant for a enzyme) aka the Michaelis constant (~equal to (Kr/Kf) which is Kd)
Equation is similar to equation for fraction of ligand bound to protein receptor and both equations give a hyperbolic curve.
The basic assumptions made in Michaelis Menten model assume reversible binding off the ligand (substrate) to enzyme - so equations look similar.
Vmax can be thought of as the reaction rate when ALL binding sites on ALL enzyme molecules are occupied by substrate. In this simplistic view Km =Kd the dissociation constant for ES complex
Meaning and significance of the parameters in the Michaelis Mentin equation
1) hyperbola
When [S]«Km, [S] + Km ≈ [S]
And Vo ≈ Vmax so rate is independent of [S]
2) linear
At constant [S], [S]/([S] + Km) is constant
& Vo= Vmax (constant)
But Vmax = Kcat[E] & therefore Vo=Kcat[E]
I.e. Vo is linearly dependent on [E]
Km and Vmax can be estimated graphically by a double reciprocal (Lineweaver burke) plot
Initial rate of reaction (V) is measured at different substrate concentrations [S]. A plot of 1/v Vs 1/[S] is a straight line with slope and intercepts.
Other more accurate graphical methods are used (Eadie Hofstee and Hanes-Woolf) but double reciprocal plots remain the clearest method to visualise enzyme kinetic data.
However, become problematic with the introduction of statistics and calculated error bars - extremely sensitive to error at lowest substrate. So better to fit data using curve fitting programme like mycurvefit.com
Kcat Vs Km meaning
Kcat is the rate constant for production of product (P) by a single enzyme molecule
Kcat[E]=Vmax
Km is the Michaelis constant:
~1/Keq= ~Kd
Kcat/Km is the efficiency of an enzyme catalysed reaction and can be compared with rate constant Knon of the uncatalysed reaction
