Enzyme Mechanics

Acid–Base Catalysis

• partial proton transfer from a Brønsted acid (a species that can donate protons; lowers the free energy of a reaction’s transition state – Eg:

• Acid catalyzed Keto-enol tautermerization • Mutarotation of glucose Is Catalyzed by Acids and by Bases

• The side chains of the amino acid residues Asp, Glu, His, Cys,Tyr, and Lys have pK’s in or near the physiological pH range which permits them to act in the enzymatic capacity of general acid and/or base catalysts

• Catalysing reactions - Hydrolysis of peptides and esters, the reactions of phosphate groups, tautomerizations, and additions to carbonyl groups

RNase A Reaction

• This digestive enzyme functions to hydrolyze RNA to its component nucleotides

• The RNase A reaction exhibits a pH rate profile that peaks near pH 6

• RNase A has two essential His residues, His 12 and His 119, which act in a concerted manner as general acid and base catalysts

• the RNase A reaction is a two-step process

• His 12, acting as a general base, abstracts a proton from an RNA 2.-OH group, thereby promoting its nucleophilic attack on the adjacent phosphorus atom. Meanwhile, His 119, acting as a general acid, promotes bond scission by protonating the leaving group

• The 2.,3.-cyclic intermediate is hydrolyzed through what is essentially the reverse of the first step in which water replaces the leaving group. Thus His 12 acts as a general acid and His 119 as a general base to yield the hydrolyzed RNA and the enzyme in its original state

RNase A Reaction

Covalent Catalysis

• involves rate acceleration through the transient formation of a catalyst–substrate covalent bond

– Eg:

• decarboxylation of acetoacetate

• Covalent Catalysis Has Both Nucelophilic and Electrophilic Stages

• There are three stages

1. The nucleophilic reaction between the catalyst and the substrate to form a covalent bond.

2. The withdrawal of electrons from the reaction center by the now electrophilic catalyst.

3. The elimination of the catalyst, a reaction that is essentially the reverse of stage 1.

• Reaction mechanisms can either begin with nucleophilic catalysis or electrophilic catalysis depending on which of these effects provides the greater driving force for the reaction(rate-determining step)

• An important aspect of covalent catalysis is that the more stable the covalent bond formed, the less facilely it will decompose in the final steps of a reaction

• A good covalent catalyst must therefore combine the seemingly contradictory properties of high nucleophilicity and the ability to form a good leaving group, that is, to easily reverse the bond formation step

• imidazole and thiol functions (highly mobile electrons) have these properties

• Certain Amino Acid Side Chains and Coenzymes can Serve as Covalent Catalysts

• imidazole moiety of His, the thiol group of Cys, the carboxyl function of Asp, and the hydroxyl group of Ser

• several coenzymes,

• most notably thiamine pyrophosphate and pyridoxal phosphate, function in association with their apoenzymes mainly as covalent catalysts

Metal Ion Catalysis

• Nearly one-third of all known enzymes require the presence of metal ions for catalytic activity

• There are two classes of metal ion–requiring enzymes that are distinguished by the strengths of their ion–protein interactions

• 1. Metalloenzymes contain tightly bound metal ions, most commonly transition metal ions such as Fe2, Fe3, Cu2, Zn2,Mn2, or Co3.

• 2. Metal-activated enzymes loosely bind metal ions from solution, usually the alkali and alkaline earth metal ions Na, K,Mg2, or Ca2.

• Metal ions participate in the catalytic process in three major ways:

• By binding to substrates so as to orient them properly for reaction

• By mediating oxidation–reduction reactions through reversible changes in the metal ion’s oxidation state

• By electrostatically stabilizing or shielding negative charges

Metal Ions Promote Catalysis through Charge Stabilization

• In many metal ion–catalyzed reactions, the metal ion acts in much the same way as a proton to neutralize negative charge, that is, it acts as a Lewis acid

• metal ions are often much more effective catalysts than protons because metal ions can be present in high concentrations at neutral pH’s and can have charges greater than 1

• decarboxylation of dimethyloxaloacetate, as catalyzed by metal ions such as Cu2 and Ni2 (non enzymatic example)

• In that metal ion electrostatically stabilizes the developing enolate ion of the transition state

• Most enzymes that decarboxylate oxaloacetate require a metal ion for activity.

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