[6] Statistical analysis of enzyme kinetic data

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3–8.1 to values for H44R/H48Q ADH of 8.0–9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD⁺ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/E_tK_m) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Climate change is driving a search for environmentally safe methods to produce chemicals used in ordinary life. One such molecule is 3-hydroxypropionic acid, which is a platform industrial chemical used as a precursor for a variety of other chemical end products. The biosynthesis of 3-hydroxypropionic acid can be achieved in recombinant microorganisms via malonyl-CoA reductase in two separate reactions. The reduction of malonyl-CoA by NADPH to form malonic semialdehyde is catalyzed in the C-terminal domain of malonyl-CoA reductase, while the subsequent reduction of malonic semialdehyde to 3-hydroxypropionic acid is accomplished in the N-terminal domain of the enzyme. A new assay for the reverse reaction of the N-terminal domain of malonyl-CoA reductase from Chloroflexus aurantiacus activity has been developed. This assay was used to determine the kinetic mechanism and for isotope effect studies. Kinetic characterization using initial velocity patterns revealed random binding of the substrates NADP⁺ and 3-hydroxypropionic acid. Isotope effects showed substrates react to give products faster than they dissociate and that the products of the reverse reaction, NADPH and malonic semialdehyde, have a low affinity for the enzyme. Multiple isotope effects suggest proton and hydride transfer occur in a concerted fashion. This detailed kinetic characterization of the reaction catalyzed by the N-terminal domain of malonyl-CoA reductase could aid in engineering of the enzyme to make the biosynthesis of 3-hydroxypropionic acid commercially competitive with its production from fossil fuels.

Alcohol dehydrogenase catalyzes the reversible transfer of a hydride directly from an alcohol to the nicotinamide ring of NAD⁺ to form an aldehyde and NADH, and the proton from the alcohol probably is transferred through a hydrogen-bonded system to the imidazole of His-48. Studies of the pH dependencies, and solvent and substrate isotope effects on the wild-type and the enzyme with His-48 substituted with Gln-48 were used to demonstrate a role for the proton relay system. The H48Q substitution increases affinities for NAD⁺ and NADH by ∼2-fold, suggesting that the overall protein structure is maintained. In contrast, catalytic efficiencies (V/K_m) on ethanol and acetaldehyde and affinity for 2,2,2-trifluoroethanol are decreased by about 10-fold. The pH dependencies for catalytic efficiencies on ethanol and acetaldehyde (log V/K_m versus pH), show pK values of about 7.5 for wild-type enzyme, but ethanol oxidation by H48Q ADH is essentially linear over the pH range from 5.5 to 9.2 with a slope of 0.47. Steady-state kinetics and substrate isotope effects suggest that the kinetic mechanism of H48Q ADH has become partly random for oxidation of ethanol. Both wild-type and H48Q ADHs have pH-independent isotope effects for oxidation (V₁/K_b) of 1-butanol/1-butanol-d₉ of 4, suggesting that hydride transfer is a major rate-limiting step. The pH dependence for butanol oxidation by wild type ADH shows a wavy profile over the pH range from pH 6 to 10, with a ∼2.3-fold larger V₁/K_b in D₂O than in H₂O, an “inverse” isotope effect. The substrate isotope effect of 4 is not altered by the solvent isotope effect, suggesting concerted proton/hydride transfer. The solvent isotope effect can be explained by a ground state with a water bound to the catalytic zinc in the enzyme-NAD⁺ complex, and a transition state that resembles a complex with NADH and aldehyde. In contrast, the H48Q enzyme has a diminished inverse solvent isotope effect of ∼1.3 and an essentially linear pH dependence with a slope of log V₁/K_b against pH of 0.49 for oxidation of 1-butanol, which together are consistent with a transition state where hydroxide ion directly accepts a proton from the 2′-hydroxyl group of the nicotinamide ribose in the proton relay system in the enzyme-NAD⁺-alcohol complex. The results support a catalytic role for His-48 in the proton relay system.

His-48 in yeast alcohol dehydrogenase I (His 51 in horse liver alcohol dehydrogenase) is a highly conserved residue in the active sites of many alcohol dehydrogenases. The imidazole group of His-48 may participate in base catalysis of proton transfer as it is linked by hydrogen bonds through the 2′-hydroxyl group of the nicotinamide ribose and the hydroxyl group of Thr-45 to the hydroxyl group of the alcohol bound to the catalytic zinc. In this study, His-48 was substituted with a glutamic acid residue to determine if a carboxylate could replace imidazole or to a serine residue to determine if the exposure of the 2′-hydroxyl group of the ribose to solvent would allow proton transfer to water without base catalysis. At pH 7.3, the H48E substitution increases affinity for NAD⁺ and NADH 17- or 2.6-fold, but decreases catalytic efficiency (V/K_m) on ethanol by 70-fold and on acetaldehyde by 6-fold relative to wild-type enzyme. The H48S substitution increases affinity for coenzymes by 2-fold and decreases (V/K_m) on ethanol and acetaldehyde only by ∼3-fold. The substituted enzymes show substrate deuterium isotope (H/D) effects of 3–4 for turnover number (V₁) and catalytic efficiency (V₁/K_b) for ethanol oxidation, indicating that hydrogen transfer is partially rate-limiting and suggesting a somewhat more random mechanism for binding of ethanol and NAD⁺. For reduction of acetaldehyde, the deuterium isotope effects are small, and the kinetic mechanism appears to be ordered for binding of NADH first and acetaldehyde next. The pH dependencies for H48E and H48S ADHs can be described by a mechanism with pK values of about 6–7 and 9. However, the pH dependencies for oxidation of ethanol and butanol by the H48S enzyme are also simply described by a straight line, with slopes of log V₁/K_b against pH of 0.37 or 0.43, respectively. The linear dependence apparently represents catalysis by hydroxide that has a low activity coefficient due to the protein environment, or to a kinetically complex proton transfer. The effects of the substitutions of His-48 show that this residue contributes to catalysis, although many dehydrogenases also have other residues.

Icaritin is a clinically effective and safe drug candidate for treating various cancers; however, the efficient and clean production remains challenging. Herein, a novel α-L-rhamnosidase (Rhase-I) was discovered from Talaromyces stollii CLY-6. Comprehensive studies showed that the Rhase-I can efficiently cleave both the outer and inner rhamnosidic bonds of epimedin C with the highest hydrolytic activity ever reported towards the rhamnosidic linkage between rhamnose and aglycone (13.52 U/mg and 179.67 mM⁻¹ s⁻¹ against icariin). Remarkably, the highest icaritin productivity 93.16 g/L/h/g of Rhase-I was achieved by applying Rhase-I together with a glucosidase (Bglsk) into an optimized two-step catalysis process. This enzymatic technology allows icaritin production to proceed under milder conditions (30–45 °C) with less environmental issues (no acid/base consumption), shorter duration (1 h), and higher production efficiency (93.16 g/L/h/g of Rhase-I), which offers a great opportunity for industrial clean icaritin production.

X-Ray crystallography shows that the hydroxyl group of Thr-45 in the fermentative alcohol dehydrogenase (ADH1) from Saccharomyces cerevisiae is hydrogen-bonded to the hydroxyl group of the alcohol bound to the catalytic zinc and is part of a proton relay system linked to His-48. The contribution of Thr-45 to catalysis was studied with steady state kinetics of the enzyme with the T45G substitution. Affinities for coenzymes decrease by only 2–4-fold, but the turnover numbers (V/E_t) and catalytic efficiencies (V/K_mE_t) decrease 480-fold and 2900-fold for the oxidation of ethanol and 450-fold and 8400-fold for acetaldehyde reduction, respectively, relative to wild-type enzyme. Binding of NADH appears to require protonation of a group with a pK value of ∼7.4 in wild-type ADH1, but the pK value for T45G ADH1 appears to be less than 5. For wild-type enzyme, the pH dependencies for ethanol oxidation (V₁/E_t and V₁/K_bE_t) are maximal above pK values of 7.0–7.7 and are attributed to the ionization of the alcohol or water bound to the catalytic zinc facilitated by His-48 in the enzyme-NAD⁺ complexes. For T45G ADH1, these pK values are shifted to 6.3. The reduction of acetaldehyde (V₂/E_t and V₂/K_pE_t) modestly increases as the pH increases for wild-type and T45G enzymes. The removal of the hydroxyethyl group of Thr-45 disrupts the connection of the oxygen of ligands bound to the catalytic zinc with the proton relay system and formation of productive catalytic states. The conformational change of the enzyme and the exchange of ligands on the catalytic zinc can also be affected. Assignments of groups responsible for the pK values are discussed in the context of studies on other forms of horse liver and yeast ADHs. The substitutions with Ala-45 and Cys-45 in yeast ADH1 and the homologous substitutions with Ala-48 in horse and human liver ADHs also significantly decrease catalytic efficiency. Threonine or serine residues at this position in alcohol dehydrogenases are highly conserved and contribute substantially to catalysis.