chapter 6- mechanism of enzymes

what are the principles of enzyme catalysis?

• covalent interactions

• non-covalent interactions

what is the difference between covalent and non-covalent interactions?

• covalent interactions between enzyme and substrate may lower the energy of activation by providing an alternative, lower energy pathway

• non-covalent interactions are used to establish binding interactions that contribute to catalysis and transient interactions that specifically stabilize the transition state

what happens to a substrate when there is no enzyme?

using the analogy of a stick

• the stick is the substrate. it must bend into the transition state (a bent stick) before breaking into products

• there is a high activation energy meaning that the reaction is slow and inefficient 

what happens when the enzyme is complementary to substrate?

• an enzyme is tightly bound to the substrate using non-covalent interactions

• the tight fit does not help the substrate reach the transition state

• Instead of lowering activation energy, it will increase

what happens when an enzyme is complementary to transition state?

• the enzyme will loosely bind to the substrate

• it will allow the substrate to go the transition state as it is shaped to tightly bind to the TS, also using non-covalent interactions

• his is faster and efficient and will lower the activation energy

what does a 10-fold increase correspond to?

• 5.7 kJ/mol of stabilization

what does a 100-fold correspond to?

• it is 5.7kJ/mol * 2

• so it is 11.4kJ/mol

what does a 10¹⁷ fold correspond to?

• it is 5.7kJ/mol * 17

• so it is 96.9kJ/mol

why are weak interaction important in catalysis?

• energy available from a single weak interaction is 4-30 kJ/mol

• so a few weak interactions that are position correctly can achieve significant catalysis

why do enzyme active sites exclude water?

• water competes with substrate for hydrogen bonding and can disrupt the precise network of weak interactions

explain how this reaction relates to catalysis

• in this reaction, the phosphoryl group plays a role in positioning the substrate for catalysis

• these phosphoryl group interactions account for more than 80% of rate acceleration and help to discriminate between different substrates

what mechanisms (non-covalent interactions) help with catalysis?

• entropy reduction → locks the substrate in place to be correctly oriented

• desolvation of substrate → enzyme binding pushes out water allowing direct contact between enzyme and substrate

• rearrangement of substrate → nudging it toward the transition state

• conformation change in enzyme → change shape when substrate binds

where is the formation of an ES complex placed?

• in proximity to reactive amino acid residues in the enzyme active site

what are the two major chemical modes of catalysis?

• acid-base catalysis

• covalent catalysis

how are amino acids residues important in

• they are involved in catalysis

• can help with the binding of substrates and transitions states

• His is 6x more likely to be involved as its side chain can act as proton donor or acceptor

in acid-base catalysis, how is acceleration of a reaction achieved?

• by catalytic transfer of a proton

what is the difference between general acid-base and specific acid-base catalysis?

• general acid base:

  • proton transferring agents

  • often an amino acid side chain

• specific acid-base

  • uses H⁺ or OH^-

what are the two ways that a proton acceptor can assist reactions?

• can cleave O-H, N-H, and even some C-H bonds by removing a proton

• general base can participate in cleavage of other bonds involving carbon (C-N bonds) by generating the equivalent of OH^- in neutral solution through removal of a proton from a molecule of water

how does metal ion catalysis contribute to catalysis?

• they have a preference in geometry

• can stabilize negative charges

• lower pka of water molecules for nuc attacks

explain what is happening in this reaction

• enzyme is using Zn to polarize a carbonyl group (stabilizes neg charge) and Glu to activate water for nuc attack (gen base)

• substrate undergoes nuc attack to form intermediate

what happens in covalent catalysis?

• substrate is bound covalently to the enzyme to form a reactive intermediate

• nucleophilic catalysis is more common

what is an important property of enzymes?

• they have the ability to couple reactions

• new pathway for lower activation energy

why is the serine protease mechanism important?

• understand how chymotrypsin works

  • cleaves peptide bonds adjacents to aromatic amino acids (Tyr, Trp, Phe)

• illustrates the principle of transition-state stabilization

• illustrates the use of general acid-base catalysis and covalent catalysis

what is something special about serine proteases?

• they can cleave simple organic esters

what does it mean for serine protease to display “burst” kinetics?

• rapid release of product followed by a slower, steady rate

• suggests that the enzyme quickly forms a covalent intermediate with the substrate

  • breakdown of that intermediate is slower

• first product is released fast while second product is released slow

what are the highlights of the serine protease mechanism?

• it is a mixture of covalent and general acid-base catalysis

• Asp¹⁰² functions only to orient His⁵⁷

• His⁵⁷ acts as a general acid and base

• Ser¹⁹⁵ forms a covalent bond with peptide to be cleaved

• covalent bond formation turns a trigonal C into a tetrahedral C

• tetrahedral oxyanion intermediate is stabilized by amid protons of Gly¹⁹³ and Ser¹⁹⁵

what is the catalytic triad in serine protease?

• Asp → stabilizes His

• Ser → nuc

• His → general acid/base

describe the first step of hydrolytic cleavage of a peptide bond by chymotrypsin

• chymotrypsin is the free enzyme

• it has an active site (very similar in all the serine proteases)

• it has a hydrophobic pocket (interactions specially bind Tyr, Phe, Trp)

• substrate binds → side chain of the residue adjacent to the peptide bond to be cleaved goes in a hydrophobic pocket on the enzyme, which positions the PB for attack

Once step 1 occurs, what is formed?

• an ES complex

• the substrate is in a hydrophobic pocket

• the interaction between His⁵⁷ and Ser¹⁹⁵ created a strong nuc alkoxide ion on Ser¹⁹⁵

• ion attack the peptide carbonyl group (short lived negative charge on the carbonyl oxygen of the substrate, which stabilized by HB in oxyanion hole)

• think about the OH donating pair to C=O and the double bond donating to O… HB happens

what is step 2 of Hydrolytic Cleavage of a Peptide Bond by Chymotrypsin?

• there is a short-lived intermediate

• there is instability of neg charge on the substrate bc of the HB

• reformation of double bond with C displaces the bond between amino group of peptide linakge… PEPTIDE BREAKAGE

• amino group is protonated by His⁵⁷ 

• NOT the intermediate with appreciable lifetime

what is step 3 of Hydrolytic Cleavage of a Peptide Bond by Chymotrypsin?

• the first product is made: part of the substrate that was pronated by the His⁵⁷

• Leaving an acyl enzyme intermediate

• It leaves the intermediate responsible for the steady state velocity

what is step 4 of Hydrolytic Cleavage of a Peptide Bond by Chymotrypsin?

• incoming water molecule is deprotonated by general base catalysis → strong nuc hydroxide ion

• attack of hydroxide on ester linkage → neg charge in oxyanion hole

what is step 5 of Hydrolytic Cleavage of a Peptide Bond by Chymotrypsin?

• collapse to form second product

• neg charge goes away by reformation of double bond then O on the other side is LG

• displacement of Ser¹⁹⁵

what is step 6 of Hydrolytic Cleavage of a Peptide Bond by Chymotrypsin?

• the enzyme-product 2 complex!!

what is step 7 of Hydrolytic Cleavage of a Peptide Bond by Chymotrypsin?

• the second product is released but its the product from the hydrophobic pocket

• leaves what we originally started with

what is interesting about serine proteases?

• they all have identical mechanisms

• they have different substrate specificity

• there is also Trysin: long, positively charges side chains

• there is also elastase: small side chains

what is subtilisin?

• bacterial serine protease with a diff overall structure

• uses the same catalytic triad

• it is non specific

• no sequence homology with chymotrypsin

what did the mutation analysis of the catalytic triaf show?

• each mutant droped down to 10^-5s^-1

  • one residue missing disrupts catalysis but some residual activity remains

• triad works as a cooperative system

why is the specificity pocket important?

• it determines which peptide are cleaved by serine proteases

• chymotrypsin → large, hydrophobic for tyr

• trypsin → narrow, negatively charged for arg

• elastase → small, hydrophobic for ala

what are other classes of proteases?

• cysteine proteases

• aspartyl proteases

• metalloproteases

Describe cysteine proteases

• residue activated by histidine residue plays a role of the nuc that attacks the peptide bond

• better nuc compared to serine proteases

• only has cys and his

• happens in papain, cathepsins, and caspases

Describe aspartyl proteases

• pair of aspartic acid residues act together to allow a water molecule to attack the peptide bond