Bound Enzymes

Fig. 6. Diagram of an immobilized enzyme electrode, where S is the substrate and P is the enzyme-bound substrate product.

The substrate concentration when the half maximal rate, (Vmax/2), is achieved is called the Km. For many simple reactions it can easily be shown that the Km is equal to the dissociation constant, Kd, of the ES complex. The Km, therefore, describes the affinity of the enzyme for the substrate. For more complex reactions, Km may be regarded as the overall dissociation constant of all enzyme-bound species. 

The addition of COg to ribulose-l,5-bisphosphate results in the formation of an enzyme-bound intermediate, 2-carboxy,3-keto-arabinitol (Figure 22.24). This intermediate arises when COg adds to the enediol intermediate gener-... 

Squalene monooxygenase, an enzyme bound to the endoplasmic reticulum, converts squalene to squalene-2,3-epoxide (Figure 25.35). This reaction employs FAD and NADPH as coenzymes and requires Og as well as a cytosolic protein called soluble protein activator. A second ER membrane enzyme, 2,3-oxidosqualene lanosterol cyclase, catalyzes the second reaction, which involves a succession of 1,2 shifts of hydride ions and methyl groups. 

Enzymes function through a pathway that involves initial formation of an enzyme-substrate complex E S, a multistep chemical conversion of the enzyme-bound substrate into enzyme-bound product E - P, and final release of product from the complex. 

Steps 3-4 of Figure 29.2 Hydrolysis The second nucleophilic acyl substitution step hydrolyzes the acyl enzyme and gives the free fatty acid by a mechanism analogous to that of the first two steps. Water is deprotonated by histidine to give hydroxide ion, which adds to the enzyme-bound acyl group. The tetrahedral... 

The retro-Claisen reaction occurs by initial nucleophilic addition of a cysteine -SH group on the enzyme to the keto group of the /3-ketoacyl CoA to yield an alkoxide ion intermediate. Cleavage of the C2-C3 bond then follows, with expulsion of an acetyl CoA enolate ion. Protonation of the enolate ion gives acetyl CoA, and the enzyme-bound acyl group undergoes nucleophilic acyl substitution by reaction with a molecule of coenzyme A. The chain-shortened acyl CoA that results then enters another round of tire /3-oxidation pathway for further degradation. 

Step 3 of Figure 29.11 Reaction with Lipoamide Hydroxyethylthiamin diphosphate is an enamine (R2N—C=C), which, like all enamines, is nucleophilic (Section 23.11). It therefore reacts with the enzyme-bound disulfide lipoamide by nucleophilic attack on a sulfur atom, displacing the second sulfur in an SN2-like process. 

Q An amino acid reacts with the enzyme-bound PLP inline by nucleophilic addition of its -NH2 group to the C=N bond of the imine, giving a PLP-amino acid imine and releasing the enzyme amino group. 

It is likely that the madurastatins are biosynthesized on a nonribosomal peptide synthetase, from salicylic acid as the starter acid. L-Serine is probably the precursor to the aziridine moiety, with epimerization occurring on the enzyme-bound amino acid as found for other nonribosomal peptides, with aziridine formation occurring at a late stage. Compounds 120 and 123 could therefore be biosynthetic precursors to 119 and 122, respectively. 

Biotin can be synthesized by the human colon flora. The question to which extent this production contributes to covering the host-organism s requirements is, however, subject to discussion. In most foods of animal origin as well as in cereals, biotin prevails in the protein (= enzyme)-bound form as e-N-biotinyl-L-lysine (= biocytin). Brewer s yeast, liver, soya beans, and peanuts number among the biotin rich foods [1]. 

In nature, aminotransferases participate in a number of metabolic pathways [4[. They catalyze the transfer of an amino group originating from an amino acid donor to a 2-ketoacid acceptor by a simple mechanism. First, an amino group from the donor is transferred to the cofactor pyridoxal phosphate with formation of a 2-keto add and an enzyme-bound pyridoxamine phosphate intermediate. Second, this intermediate transfers the amino group to the 2-keto add acceptor. The readion is reversible, shows ping-pong kinetics, and has been used industrially in the production ofamino acids [69]. It can be driven in one direction by the appropriate choice of conditions (e.g. substrate concentration). Some of the aminotransferases accept simple amines instead of amino acids as amine donors, and highly enantioselective cases have been reported [70]. 

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... 

Substrate analogs which promise to be particularly good active-site probes are those which are conformationally restricted. One key feature of enzymatic processes is that when a substrate is bound to an enzyme, probably only one of the many possible conformations of the substrate molecule is assumed. Consequently, before a detailed mechanism for an enzymatic process can be formulated, the preferred conformations of each of the enzyme-bound substrates must be known. ... 

In subsequent experiments (66), this locked substrate was used to obtain evidence for the hypothesis (67) that enzyme-bound y-glutamyl phosphate 14 is an intermediate in the enzyme-catalyzed reaction. All attempts to isolate this acyl phosphate 14 have failed (66), presumably because of the marked tendency of this intermediate to cyclize to pyrrolidonecarboxyUc acid, 15, and to hydrolyze to glutamic acid. 

In other cases, new asymmetric centers may be built into the substrate so that the stereochemical course of the overall reaction may be elucidated. The preferred conformation of the natural substrate when bound to the enzyme may be deduced and regions in the space around the enzyme-bound substrate where substituents can be tolerated may be inferred. 

A transition to irreversible inhibitors is seen in pseudosubstrates forming enzyme-bound intermediates which are cleaved very slowly, for example, the 2-deoxy-2-fluoro-D-glycosides to be discussed in Section II,3,b. 

Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially, oxalosucci-nate, which remains enzyme-bound and undergoes decarboxylation to a-ketoglutarate. The decarboxylation... 

The P-alanyl dipeptides carnosine and anserine (A -methylcarnosine) (Figure 31-2) activate myosin ATPase, chelate copper, and enhance copper uptake. P-Alanyl-imidazole buffers the pH of anaerobically contracting skeletal muscle. Biosynthesis of carnosine is catalyzed by carnosine synthetase in a two-stage reaction that involves initial formation of an enzyme-bound acyl-adenylate of P-alanine and subsequent transfer of the P-alanyl moiety to L-histidine. 

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