Enzymes lyases

Lyases. These enzymes cleave C-C, C-0, C-N and other bonds by elimination leaving double bonds or conversely add groups to double bonds. This group includes decarboxylases, hydratases, dehydratases and some carboxylases. 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

Biosynthesis of Tea Flavonoids. The pathways for the de novo biosynthesis of flavonoids in both soft and woody plants (Pigs. 3 and 4) have been generally elucidated and reviewed in detail (32,51). The regulation and control of these pathways in tea and the nature of the enzymes involved in synthesis in tea have not been studied exhaustively. The key enzymes thought to be involved in the biosynthesis of tea flavonoids are 5-dehydroshikimate reductase (52), phenylalanine ammonia lyase (53), and those associated with the shikimate/arogenate pathway (52). At least 13 enzymes catalyze the formation of plant flavonoids (Table 4). 

One of the most interesting uses for cinnamic acid in recent years has been as a raw material in the preparation of L-phenylalanine [63-91-2] the key intermediate for the synthetic dipeptide sweetener aspartame (25). Genex has described a biosynthetic route to L-phenylalanine which involves treatment of immobilized ceUs of R rubra containing the enzyme phenylalanine ammonia lyase (PAT,) with ammonium cinnamate [25459-05-6] (26). 

In the first edition of this book this chapter was entitled "Antiparallel Beta Structures" but we have had to change this because an entirely unexpected structure, the p helix, was discovered in 1993. The p helix, which is not related to the numerous antiparallel p structures discussed so far, was first seen in the bacterial enzyme pectate lyase, the stmcture of which was determined by the group of Frances Jurnak at the University of California, Riverside. Subsequently several other protein structures have been found to contain p helices, including extracellular bacterial proteinases and the bacteriophage P22 tailspike protein. 

Figure 5.30 Schematic diagrams of the structure of the enzyme pectate lyase C, which has a three-sheet parallel P-helix topology.

Finally, citrate can be exported from the mitochondria and then broken down by ATP-citrate lyase to yield oxaloacetate and acetyl-CoA, a precursor of fatty acids (Figure 20.23). Oxaloacetate produced in this reaction is rapidly reduced to malate, which can then be processed in either of two ways it may be transported into mitochondria, where it is reoxidized to oxaloacetate, or it may be oxidatively decarboxylated to pyruvate by malic enzyme, with subse-... 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

Enzymes are classified into six categories depending on the kind of reaction they catalyze, as shown in Table 26.2. Oxidoreductases catalyze oxidations and reductions hansferases catalyze the transfer of a group from one substrate to another hydrolases catalyze hydrolysis reactions of esters, amides, and related substrates lyases catalyze the elimination or addition of a small molecule such as H2O from or to a substrate isomerases catalyze isomerizalions and ligases catalyze the bonding together of two molecules, often coupled with the hydrolysis... 

The first step in the biological degradation of histidine is formation of a 4-methylideneimidazol-5-one (MIO) by cyclization of a segment of the peptide chain in the histidine ammonia lyase enzyme. Propose a mechanism. 

Enzymes such as lyases, transferases and isomerases (Table 2.1) account for most of the remainder of industrially applied biotransformations. 

Reaction 1 is governed by the enzyme phenylalanine ammonia lyase. This enzyme normally conducts the breakdown of L-phenylalanine to from-cinnamic add and ammonia. However, die reaction can be reversed leading to the production of L-phenylalanine from frans-dnnamic add by using excess ammonia. 

With dimethylmalic acid lyase (EC 4.1.3.32) from Clostridium harkeri. an enzyme involved in nicotinic acid metabolism, propanoate can be added to pyruvate yielding stereospecifically the (2i ,3S)-dimethylmalic acid39. 

A subclass of lyases, involved in amino acid metabolism, utilizes pyridoxal 5-phosphate (PLP, 3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]-4-pyridinecarbaldehyde) as a cofactor for imine/ enamine-type activation. These enzymes are not only an alternative to standard fermentation technology, but also offer a potential entry to nonnatural amino acids. Serine hydroxymethyl-tansferase (SHMT EC 2.1.2.1.) combines glycine as the donor with (tetrahydrofolate activated) formaldehyde to L-serine in an economic yield40, but will also accept a range of other aldehydes to provide /i-hydroxy-a-amino acids with a high degree of both absolute and relative stereochemical control in favor of the L-erythro isomers41. 

Resistance to streptogramin type B antibiotics can be mediated in staphylococci and enterococci by plasmids carrying a vgb gene [2]. The Vgb enzyme is a lyase that linearizes the cyclic hexadepsipeptide by cleavage of the ester bond via an elimination reaction. 

CYP17 is the 17 alpha-hydroxylase and 17-20 lyase, two different reactions catalyzed by one enzyme and required for production of testosterone and estrogen, respectively. Defects in this enzyme affect development at puberty. 

Typically, lyases are quite specific for the nucleophilic donor component owing to mechanistic requirements. Usually, approach of the aldol acceptor to the enzyme-bound nucleophile occurs stereospedfically following an overall retention mechanism, while the facial differentiation of the aldehyde carbonyl is responsible for the relative stereoselectivity. In this manner, the stereochemistry of the C—C bond formation is completely controlled by the enzymes, in general irrespective of the constitution or configuration of the substrate, which renders the enzymes highly predictable. On the other hand, most of the lyases allow a reasonably broad variation of the electrophilic acceptor component that is usually an aldehyde. This feature... 

N-Acetylneuraminic acid aldolase (or sialic acid aldolase, NeuA EC 4.1.3.3) catalyzes the reversible addition of pyruvate (2) to N-acetyl-D-mannosamine (ManNAc (1)) in the degradation of the parent sialic acid (3) (Figure 10.4). The NeuA lyases found in both bacteria and animals are type I enzymes that form a Schiff base/enamine intermediate with pyruvate and promote a si-face attack to the aldehyde carbonyl group with formation of a (4S) configured stereocenter. The enzyme is commercially available and it has a broad pH optimum around 7.5 and useful stability in solution at ambient temperature [36]. 

Comparable to the situation for the NeuA and KdoA enzyme pair (see above), a class I lyase complementary to the KDPGIc aldolase that has a stereopreference for the (4S)-configuration is known (Figure 10.11). The aldolase, which acts on... 

A biochemically related benzaldehyde lyase (BAL) (EC 4.1.2.38) catalyzes the same carboligation reactions, but with opposite (J )-selectivity (mf-110) [178]. All these enzymes seem to display a rather useful substrate tolerance for variously substituted aldehyde precursors. 

Figure 7-5. Two-dimensional representation of Koshland s induced fit model of the active site of a lyase. Binding of the substrate A—B induces conformational changes In the enzyme that aligns catalytic residues which participate in catalysis and strains the bond between A and B, facilitating its cleavage.

Pymvate dehydrogenase is a mitochondrial enzyme, and fatty acid synthesis is a cytosohc pathway, but the mitochondrial membrane is impermeable to acetyl-CoA. Acetyl-CoA is made available in the cytosol from citrate synthesized in the mitochondrion, transported into the cytosol and cleaved in a reaction catalyzed by ATP-citrate lyase. 

Both dehydrogenases of the pentose phosphate pathway can be classified as adaptive enzymes, since they increase in activity in the well-fed animal and when insulin is given to a diabetic animal. Activity is low in diabetes or starvation. Malic enzyme and ATP-citrate lyase behave similarly, indicating that these two enzymes are involved in lipogenesis rather than gluconeogenesis (Chapter 21). 

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