The allosteric modulation of lipases and its possible biological relevance

Abstract

Background: During the development of an enantioselective synthesis using the lipase from Mucor miehei an unusual reaction course was observed, which was analyzed precisely. For the first time an allosteric modulation of a lipase changing its selectivity was shown.

Theory: Considering the biological relevance of the discovered regulation mechanism we developed a theory that describes the regulation of energy homeostasis and fat metabolism.

Conclusion: This theory represents a new approach to explain the cause of the metabolic syndrome and provides an innovative basis for further research activity.

Figure 1

Synthesis of the prochiral diol 3.

Figure 2

Lipase catalyzed enantioselective, irreversible acetylation.

Figure 3

Lipase catalyzed acetylation of the prochiral diol 3.

Figure 4

Enantioselectivity of both lipases (lipase from B. cepacia and from M. miehei).

Figure 5

Progress of the reaction carried out at +20°C; a, c, e: Amount of compounds 3, 4 and 5 (n [%]); b, d, f: Enantiomeric excess of (S)-4 (% ee); a, b: Transformation catalyzed by lipase from B. cepacia; c, d: Transformation catalyzed by lipase from M. miehei; e, f: Simulation of the reaction using a constant lipase activity a = 0.004 [14]; The rate constants k₁ to k₈ are defined in Figure 3; k₁ = 15, k₂ = 1, k₃ = 4, k₄ = 60, k₅ = 15·10^-6, k₆ = 1·10^-6, k₇ = 4·10^-6, k₈ = 60·10^-6.

Figure 6

Progress of the reaction carried out at low temperature; a, c, e: Amount of compounds 3, 4 and 5 (n [%]); b, d, f: Enantiomeric excess of (S)-4 (% ee); a, b: Transformation catalyzed by lipase from B. cepacia at -40°C; c, d: Transformation catalyzed by lipase from M. miehei at -10°C; e, f: Simulation of the reaction using a constant lipase activity a = 0.004 [14]; The rate constants k₁ to k₈ are defined in Figure 3; k₁ = 35, k₂ = 1, k₃ = 4, k₄ = 140, k₅ = 35·10^-6, k₆ = 1·10^-6, k₇ = 4·10^-6, k₈ = 140·10^-6.

Figure 7

Possible explanations for a constant enantiomeric excess during the progress of the reaction; a: non-enantioselective transformation of (S)-4 and (R)-4 into 5; b: inhibited transformation of (S)-4 and (R)-4 into 5; c: non-competitive, since compounds 4 and 5 do not bind to the enzyme (indicated by crosses).

Figure 8

Simulated reaction courses using a constant lipase activity a [14]; The rate constants k₁ to k₈ are defined in Figure 3; a, c, e: Amount of compounds 3, 4 and 5 (n [%]); b, d, f: Enantiomeric excess of (S)-4 (% ee); a, b: non-enantioselective transformation of (S)-4 and (R)-4 into 5 (a = 0.0008); k₁ = 17, k₂ = 1, k₃ = 9, k₄ = 9, k₅ = 17·10^-6, k₆ = 1·10^-6, k₇ = 9·10^-6, k₈ = 9·10^-6; c, d: inhibited transformation of (S)-4 and (R)-4 into 5 (a = 0.0004); k₁ = 17, k₂ = 1, k₃ = 0.009, k₄ = 0.009, k₅ = 17·10^-6, k₆ = 1·10^-6, k₇ = 0.009·10^-6, k₈ = 0.009·10^-6; e, f: non-competitive, since compounds 4 and 5 do not bind to the enzyme (a = 0.02); k₁ = 17, k₂ = 1.

Figure 9

Progress of the reaction carried out at -10°C using lipase from M. miehei; Amount of compounds 3, 4 and 5 (n [%]); The reaction course is divided into two time periods: a: period A (compounds 4 and 5 do not bind to the lipase); b: time period B (the reaction is competitive).

Figure 10

Reaction schemes that explain the progress of the reaction using lipase from M. miehei (Fig. 9); a: during time period A compounds 4 and 5 do not bind to the lipase (indicated by crosses).; b: during time period B the reaction is competitive.

Figure 11

Conformation thesis of the allosteric modulation to explain the progress of the reaction using lipase from M. miehei (Fig. 9); a: during time period A compound 3 binds allosterically modifying the active binding site.; b: during time period B the allosteric modulation is reversed.

Figure 12

Structural analogy of the compounds used in the experiments (a) and natural substrates (b); solid lines encircle large lipophilic moieties (modified OH-groups), dashed lines encircle small hydrophilic moieties (free OH-groups); a: diacetate 5, monoacetate 4 and diol 3; b: triglyceride TG, diglyceride DG and 2-monoglyceride 2-MG (stearic acid as fatty acid).

Figure 13

Conformation thesis of the allosteric modulation shown with natural substrates; TG = triglyceride; DG = diglyceride; 2-MG = 2-monoglyceride; a: In case of modulation A only 2-MGs can bind to the active site and can be transformed into DGs; b: In case of modulation B all compounds can bind to the active site. TGs and DGs can be hydrolyzed.

Figure 14

The allosteric regulation is exemplarily shown by means of a membrane bound lipase. Triglycerides (TGs) and diglycerides (DG) cannot pass through the membrane, whereas monoglycerides (MGs), free fatty acids (FFAs) and glycerol (GLY) which are the products of hydrolysis can be absorbed. Due to the allosteric mechanism the lipase hydrolyzes just as many TGs and DGs as are actually needed.

Figure 15

Organisms using type-1 or type-2 lipases to hydrolyze nutritional fat; a: Type-1 lipases hydrolyze fat to a large extent. The lipase secreting organism can depart by active or passive movement.; b: Type-2 lipases are regulated allosterically, therefore they hydrolyze fat depending on the concentration of products.

Figure 16

a: Supposed endosymbiosis of an organism containing type-2 lipases. As a result the allosteric regulation mechanism controls the concentration of products of hydrolysis and fat can be accumulated. This is a mutualistic symbiosis, as the effect is beneficial to both endo- and exosymbiont.; b: A new kind of organism results from genetic combination of the symbionts. The ability to accumulate and mobilize storage fat is maintained and provides a major benefit due to energy homeostasis.

Figure 17

Possible formation of organisms upon endosymbiosis; a: An organism originating from symbiosis of an organism containing type-2 lipases and a cogenerous one is fungus-like; b: As the former exosymbiot used type-1 lipases to hydrolyze nutritional fat, an animal-like organism results.; c: If the animal-like organism does not need nutritional fat, since it can produce metabolic energy by photosynthesis, it can assemble a cell wall and becomes plant-like.

Figure 18

Schematic pathways of the human fat metabolism established until 2004. Nutritional fat is hydrolyzed by human pancreatic lipase (HPL) to produce 2-monoglycerides, glycerol and free fatty acids, which are absorbed. In addition to glucose these products provide metabolic energy directly or they can be transformed into adipose. Adipose is mobilized by hormone-sensitive lipase (HSL).

Figure 19

Scheme of the human fat metabolism as supposed according to our hypothesis. Human pancreatic lipase (HPL) hydrolyzes nutritional fat. The recently discovered adipose triglyceride lipase (ATGL) is rate limiting for the mobilization and storage of adipose. We assume that ATGL is regulated allosterically in the way we demonstrated herein. Irrespective of food supply this regulation mechanism keeps the concentration of the products of hydrolysis constant and maintains energy homeostasis. Hormone-sensitive lipase (HSL) is of minor importance.