Enzymes rate-limiting steps

The neurotransmitter must be present in presynaptic nerve terminals and the precursors and enzymes necessary for its synthesis must be present in the neuron. For example, ACh is stored in vesicles specifically in cholinergic nerve terminals. It is synthesized from choline and acetyl-coenzyme A (acetyl-CoA) by the enzyme, choline acetyltransferase. Choline is taken up by a high affinity transporter specific to cholinergic nerve terminals. Choline uptake appears to be the rate-limiting step in ACh synthesis, and is regulated to keep pace with demands for the neurotransmitter. Dopamine [51 -61-6] (2) is synthesized from tyrosine by tyrosine hydroxylase, which converts tyrosine to L-dopa (3,4-dihydroxy-L-phenylalanine) (3), and dopa decarboxylase, which converts L-dopa to dopamine. 

The dopamine is then concentrated in storage vesicles via an ATP-dependent process. Here the rate-limiting step appears not to be precursor uptake, under normal conditions, but tyrosine hydroxylase activity. This is regulated by protein phosphorylation and by de novo enzyme synthesis. The enzyme requites oxygen, ferrous iron, and tetrahydrobiopterin (BH. The enzymatic conversion of the precursor to the active agent and its subsequent storage in a vesicle are energy-dependent processes. 

Hydrolysis of esters and amides by enzymes that form acyl enzyme intermediates is similar in mechanism but different in rate-limiting steps. Whereas formation of the acyl enzyme intermediate is a rate-limiting step for amide hydrolysis, it is the deacylation step that determines the rate of ester hydrolysis. This difference allows elimination of the undesirable amidase activity that is responsible for secondary hydrolysis without affecting the rate of synthesis. Addition of an appropriate cosolvent such as acetonitrile, DMF, or dioxane can selectively eliminate undesirable amidase activity (128). 

Catecholamine biosynthesis begins with the uptake of the amino acid tyrosine into the sympathetic neuronal cytoplasm, and conversion to DOPA by tyrosine hydroxylase. This enzyme is highly localized to the adrenal medulla, sympathetic nerves, and central adrenergic and dopaminergic nerves. Tyrosine hydroxylase activity is subject to feedback inhibition by its products DOPA, NE, and DA, and is the rate-limiting step in catecholamine synthesis the enzyme can be blocked by the competitive inhibitor a-methyl-/)-tyrosine (31). 

Enzymes assist formation of proper disulfide bonds during folding Isomerization of proline residues can be a rate-limiting step in protein folding Proteins can fold or unfold inside chaperonins GroEL is a cylindrical structure with a... 

See also Enzyme cofactors downhill trajectories for, 196,197 mechanism of catalytic reaction, 190-192 metal substitution, 200-204 potential surfaces for, 192-195,197 rate-limiting step of, 190 reference solution reaction for, 192-195,... 

Reaction 1 is the slowest step in this series of reactions leading to product formation. It is the rate-limiting step. Since this reaction involves bringing E and S together, it is a second-order reaction overall and first order with respect to the total enzyme concentration and the substrate concentration. 

Hence, l/K only approximates l/K under conditions where the association and dissociation of the ES complex is rapid relative to the rate-limiting step in catalysis. For the many enzyme-catalyzed reactions for which + kj is not approximately equal to k j, IIK will underestimate IIK,. 

Testiculat androgens are synthesized in the interstitial tissue by the Leydig cells. The immediate precursor of the gonadal steroids, as for the adrenal steroids, is cholesterol. The rate-limiting step, as in the adrenal, is delivery of cholesterol to the inner membrane of the mitochondria by the transport protein StAR. Once in the proper location, cholesterol is acted upon by the side chain cleavage enzyme P450scc. The conversion of cholesterol to pregnenolone is identical in adrenal, ovary, and testis. In the latter two tissues, however, the reaction is promoted by LH rather than ACTH. 

The turnover rate of a transmitter can be calculated from measurement of either the rate at which it is synthesised or the rate at which it is lost from the endogenous store. Transmitter synthesis can be monitored by administering [ H]- or [ " C]-labelled precursors in vivo these are eventually taken up by neurons and converted into radiolabelled product (the transmitter). The rate of accumulation of the radiolabelled transmitter can be used to estimate its synthesis rate. Obviously, the choice of precursor is determined by the rate-limiting step in the synthetic pathway for instance, when measuring catecholamine turnover, tyrosine must be used instead of /-DOPA which bypasses the rate-limiting enzyme, tyrosine hydroxylase. 

Tyrosine is converted to dopa by the cytoplasmic enzyme tyrosine hydroxylase. This is the rate-limiting step 5 x 10 M) in DA synthesis, it requires molecular O2 and Fe + as well as tetrahydropterine (BH-4) cofactor and is substrate-specific. It can be inhibited by a-methyl-p-tyrosine, which depletes the brain of both DA and NA and it is particularly important for the maintenance of DA synthesis. Since the levels of tyrosine are above the for tyrosine hydroxylase the enzyme is normally saturated and so it is not possible to increase DA levels by giving tyrosine. 

Figure 8.5 The synthetic pathway for noradrenaline. The hydroxylation of the amino acid, tyrosine, which forms dihydroxyphenylalanine (DOPA) is the rate-limiting step. Conversion of dopamine to noradrenaline is effected by the vesicular enzyme, dopamine-P-hydroxylase (DpH) after uptake of dopamine into the vesicles from the cell cytosol...

Figure 9.4 The synthesis and metabolism of 5-HT. The primary substrate for the pathway is the essential amino acid, tryptophan and its hydroxylation to 5-hydrox5dryptophan is the rate-limiting step in the synthesis of 5-HT. The cytoplasmic enzyme, monoamine oxidase (MAOa), is ultimately responsible for the catabolism of 5-HT to 5-hydroxyindoleacetic acid...

Fluorouracil (5-FU) acts as a false pyrimidine, inhibiting the formation of the DNA base thymidine.26,35 The main mechanism by which it accomplishes this is by inhibiting the enzyme thymidylate synthase, the rate-limiting step in thymidine formation. 5-FU first must be metabolized to its active metabolite (F-dUMP). Additionally, metabolites of 5-FU may incorporate into RNA, inhibiting its synthesis. 

Unlike other enzymes that we have discussed, the completion of a catalytic cycle of primer extension does not result in release of the product (TP(n+1)) and recovery of the free enzyme. Instead, the product remains bound to the enzyme, in the form of a new template-primer complex, and this acts as a new substrate for continued primer extension. Catalysis continues in this way until the entire template sequence has been complemented. The overall rate of reaction is limited by the chemical steps composing cat these include the chemical step of phosphodiester bond formation and requisite conformational changes in the enzyme structure. Hence there are several potential mechanisms for inhibiting the reaction of HIV RT. Competitive inhibitors could be prepared that would block binding of either the dNTPs or the TP. Alternatively, noncompetitive compounds could be prepared that function to block the chemistry of bond formation, that block the required enzyme conformational transition(s) of turnover, or that alter the reaction pathway in a manner that alters the rate-limiting step of turnover. 

Hydrogen motion, H+, H or H, is often involved in the rate-limiting step of many enzyme catalysed reactions. Here, QM tunnelling can be important and is reflected in the values of the measured kinetic isotope effects (KIEs) [75], Enzyme motion... 

As an illustration, we briefly discuss the SCC-DFTB/MM simulations of carbonic anhydrase II (CAII), which is a zinc-enzyme that catalyzes the interconversion of CO2 and HCO [86], The rate-limiting step of the catalytic cycle is a proton transfer between a zinc-bound water/hydroxide and the neutral/protonated His64 residue close to the protein/solvent interface. Since this proton transfer spans at least 8-10 A depending on the orientation of the His 64 sidechain ( in vs. out , both observed in the X-ray study [87]), the transfer is believed to be mediated by the water molecules in the active site (see Figure 7-1). To carry out meaningful simulations for the proton transfer in CAII, therefore, it is crucial to be able to describe the water structure in the active site and the sidechain flexibility of His 64 in a satisfactory manner. 

ALAD, and ferrochelatase. Lead indirectly stimulates the mitochondrial enzyme ALAS, which catalyzes the condensation of glycine and succinyl-coenzyme A to form ALA. The activity of ALAS is the rate-limiting step in heme biosynthesis increase of ALAS activity occurs through feedback derepression. Lead... 

There are two distinct pools of HA in the brain (1) the neuronal pool and (2) the non-neuronal pool, mainly contributed by the mast cells. The turnover of HA in mast cells is slower than in neurons it is believed that the HA contribution from the mast cells is limited and that almost all brain histaminergic actions are the result of HA released by neurons (Haas Panula, 2003). The blood-brain barrier is impermeable to HA. HA in the brain is formed from L-histidine, an essential amino acid. HA synthesis occurs in two steps (1) neuronal uptake of L-histidine by L-amino acid transporters and (2) subsequent decarboxylation of l-histidine by a specific enzyme, L-histidine decarboxylase (E.C. 4.1.1.22). It appears that the availability of L-histidine is the rate-limiting step for the synthesis of HA. The enzyme HDC is selective for L-histidine and its activity displays circadian fluctuations (Orr Quay, 1975). HA synthesis can be reduced by inhibition of the enzyme HDC. a-Fluoromethylhistidine (a-FMH) is an irreversible and a highly selective inhibitor of HDC a single systemic injection of a-FMH (10-50 mg/kg) can produce up to 90% inhibition of HDC activity within 60-120 min (Monti, 1993). Once synthesized, HA is taken up into vesicles by the vesicular monoamine transporter and is stored until released. 

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