Enzyme Kinetics

Oxidoreductase class of enzymes acts as catalysts in reactions involving oxidation-reduction. Subclasses belonging to this type of enzymes include oxidase, hydroxylases, oxygenases, peroxidases, dehydrogenase, reductase, etc.

Ser and Cys residues have reactive –OH and –SH groups respectively. The compound diisopropylphosphofluoridate (DIPF) reacts with Ser residue in the active site of the enzyme acetylcholinesterase, irreversibly inhibiting the enzyme and preventing the transmission of nerve impulses. Iodoacetamide modifies Cys residues and hence, may be used as a diagnostic tool in determining whether one or more Cys residues are required for enzyme activity.

Zymogen is an enzyme’s inactive precursor which must be cleaved for the formation of active enzyme. For example, trypsin and chymotrypsin are synthesised as precursor trypsinogen and chymotrypsinogen.

E-type ATPases (stands for ‘Extracellular’) are the class of cell surface enzymes which hydrolyse NTPs like extracellular ATP and NDPs. The most likely substrates are extracellular ATP, UTP, ADP and ATP.

Phosphate translocase is second transport system. It is a symporter which transfers one H⁺ and one H₂PO₄^- into matrix. Adenine nucleotide translocase is first transport system. It is combined in inner mitochondrial membrane. It is a membrane protein (antiporter) which is used for carrying P_i and ADP into matrix and ATP into cytosol. Both  the translocases taken together are known as ATP synthasome.

DCCD is an inhibitor of F_O portion of ATP synthase. DCCD stands for Dicyclohexylcarbodiimide is an organic molecule by which protonated carboxyl groups can be covalently modified. DCCD, when combined with ATP synthase, reacts with carboxyl group of residual amino acid of subunit c at pH greater than 8 and hence, protonated at such high pH.

Addition or elimination of a proton supplied by acid/base helps to increase the substrate’s reactivity. Reaction involving two species can be accelerated by an enzyme by holding the substrate and enzyme close together. Thus, acid-base catalysis and catalysis by proximity decrease activation energy. A temporary covalent bond between substrate and enzyme is formed due to covalent catalysis. The chemical modification is temporary and the enzyme returns to the unmodified state on completion of the reaction. The substrate’s binding to enzyme’s active site weakens the target bond and make it susceptible to cleavage. Thus, covalent catalysis and catalysis by strain decrease the activation energy.

Subtilisin, a serine protease, is present in prokaryotes. Subtilisin uses a catalytic triad for creating a nucleophilic serine. Trypsins are proteases which cleave peptide bonds after arginine or lysine and this particularity is guided by the residue present at the bottom of the S1 pocket of the enzyme. Elastase proteases have a smaller S1 aperture than chymotrypsin and trypsin proteases. Therefore, residues like glycine, valine and alanine are preferred. Chymotrypsin proteases have more hydrophobic S1 pocket than trypsin proteases. Therefore, medium and large hydrophobic residues like phenylalanine, tryptophan and tyrosine are preferred. Thus, all the enzymes belong to the category of serine proteases.

Papain is a proteolytic enzyme belonging to cysteine protease family. Papain is found in unripe papaya and also in mountain papaya. Papain has a sulfhydryl group required for enzyme activity and a polypeptide chain accompanied by 3 disulfide bridges. Papain can digest protein substrates. Bromelain is a combination of proteolytic enzyme and small amounts of other substances like acid phosphatase, peroxidase, calcium and protease inhibitors. Bromelain is obtained from both stem and fruit of pineapples. Bromelain has anti-inflammatory effects and interferes with slow blood clotting.

Malonic acid is an example of competitive inhibitor because malonic acid affects succinic dehydrogenase for oxidising succinic acid to fumaric acid. The enzyme succinic dehydrogenase is prevented by malonic acid due to its structural similarity with succinic acid. Therefore, there is a competition between malonic acid and succinic acid for binding on the active site of the enzyme succinic dehydrogenase.

Tyrosinase is a rate limiting enzyme which controls melanin production. Tyrosinase is involved in the hydroxylation of monophenol and also the conversion of o-diphenol to corresponding o-quinone. The enzyme tyrosinase consists of copper and is found in animal and plant tissues. The active site of all tyrosinases contains one binuclear copper centre. Tyrosinase catalyzes melanin production and in humans it is encoded by TYR gene.

Hydrolases are those enzymes which are responsible for catalysing the substrate’s lysis with the addition of water or by hydrolysis. They help to transfer the functional group towards water. Some of the substrates are peptide, ester, halide, glycosyl, etc.

Ligases are also known as synthetases. Ligases act as catalysts in reactions where bonds are formed between two compounds. They utilise the energy obtained from cleaving the ATP. Formation of C-N, C-S, C-C, and C-O bonds are catalysed by ligases.

Competitive inhibition takes place at the enzyme’s active site. The inhibitor’s structure bears close resemblance with the structure of substrate. It forms EI complex instead of ES complex after combining with the enzyme.

Antibacterial drugs like sulpha drugs are competitive inhibitors which play an important role in the metabolism of p-amino benzoic acid or PABA. Bacteria synthesise folic acid from p-amino benzoic acid. Sulpha drugs are structurally similar to PABA and therefore sulpha drugs behave as the enzyme inhibitor and are placed at the enzyme’s active site.

The ratio K_cat/K_m is similar to the rate constant of the reaction between substrate and the enzyme. K_cat/K_m is known as the specificity constant which signifies the efficiency of an enzyme.

ATP synthase is a complex mitochondrial enzyme that is enclosed in a membrane. It acts as the catalyst to form ATP from ADP and P_i and also helps to transfer protons from positive side (intermembrane space) to the negative side (matrix) of the membrane.

Adenine nucleotide translocase is the first transport system. It is combined in inner mitochondrial membrane. It is a membrane protein (antiporter) which is used for carrying P_i and ADP into matrix and ATP into cytosol.

Dihydrofolate reductase (DHFR) uses NADPH in the form of electron donor for reducing dihydrofolic acid, a derivative of folic acid, to tetrahydrofolic acid. In humans, DHFR is encoded by DHFR gene and located in chromosome 5’s q11→q22 region. Malate dehydrogenase is a mitochondrial enzyme which is used to reduce oxaloacetate to malate in gluconeogenesis. Glycerol dehydrogenase catalyses a chemical reaction which involves 2 substrates like NAD⁺ and glycerol and 3 products like NADH, glycerone and H⁺. Structural studies of glycerol dehydrogenase have revealed that it is zinc-dependent. Tyrosinase is a rate limiting enzyme which controls melanin production. Tyrosinase is involved in the hydroxylation of monophenol and also the conversion of o-diphenol to corresponding o-quinone. The active site of all tyrosinases contains one binuclear copper centre. Serum glutamic-pyruvic transaminase, also known as alanine transaminase is present in the plasma and in different body tissues. It is commonly linked with the liver and catalyses 2 parts of alanine cycle.

Michaelis-Menten equation related to steady-state kinetics is given by

υ₀ = υ_max [S]/ (K_m + [S])
<o:p>
where υ₀ is the initial reaction rate, υ_max is the maximum reaction rate, [S] is the substrate concentration and K_m is Michaelis Constant.<o:p>