Allosteric effects in enzymes

Rob, T, GiU, P.K., Golemi-Kotra, D., Wilson, D.J. (2013) An electrospray ms-coupled microfluidic device for sub-second hydrogen/deuterium exchange pulse-labeUing reveals allosteric effects in enzyme inhibition. Lab on a Chip, 13 (13), 2528-2532. 

We observe that the solvent is arranged as a one dimensional chain of hydrogen bonded water molecules, whose properties are considerably different than ordinary three dimensional water. For example, this linear water is a better ion solvent than three dimensional water in terms of solvation energy as a function of distance from the ion to the water. In addition, the 25 A long chain of waters tends to translate as a correlated unit, thus leading us to discuss the possibility of water structures in proteins acting as the intermediary for chemical action at a distance, particularly in allosteric effects in enzymes. ... 

The cooperative binding of O2 by hemoglobin and the allosteric effects in many enzymes require interaction between sites that are widely separated in space. The MWC model was proposed in 1965 to incorporate allosteric and conformational effects in an explanation of enzyme cooperativity. The seminal observation was that most cooperative proteins have several identical subunits (protomers) in each molecule (oligomer) this situation is imperative for binding cooperativity. The MWC model is defined as follows ... 

The answer is c. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) The steps of pyrimicfine nucleotide biosynthesis are summarized in the figure below. The first step in pyrimidine synthesis is the formation of carbamoyl phosphate. The enzyme catalyzing this step, carbamoyl phosphate synthetase (1), is feedback-inhibited by UMP through allosteric effects on enzyme structure (not by competitive inhibition with its substrates). The enzyme of the second step, aspartate transcarbamoylase, is composed of catalytic and regulatory subunits. The regulatory subunit binds CTP or ATP TTP has no role in the feedback inhibition of pyrimidine synthesis. Decreased rather than increased activity of enzymes 1 and 2 would be produced by allosteric feedback inhibition. 

Although several previous reports claimed that the enzyme had been purified, Gebler and Poulter (1992) appear to have been the first to fully characterize the activity of the purified DMAT synthase. The enzyme was purified from Claviceps fusiformis ATCC 26245 [erroneously annotated in type specimen collections as a C. purpurea strain (Pazoutova and Tudzynski, 1999)]. The monomeric size was estimated at 53 kDa, and by gel filtration analysis the native enzyme was determined (at 105 kDa) to be a homodimer. Unlike other prenyltransferases, no metal ion requirement has been noted. However, when assayed in a buffer with 4 mM Ca2+, the purified protein gave a specific activity of 500 nmol/min/mg, essentially the same as with 4 mM Mg2+, but approximately twice that of the measured without added divalent cations and with the chelator EDTA included in the assay buffer. These divalent metal cations eliminated negative cooperativity of substrate binding observed both for dimethylallyl diphosphate and L-tryptophan, indicating that Ca2+ and Mg2+ probably had allosteric effects. In buffer with 4 mM MgCl2 the KM for dimethylallyl diphosphate was 8 jlM, and the KM for L-tryptophan was 12 xM. The enzyme product was authenticated by mass spectrometry, UV spectrometry, and -NMR. 

Deviations from linear behaviour usually means that the simple Michaelis-Menten mechanism is not appropriate, and curved doublereciprocal plots may indicate the presence of cooperative or allosteric effects in the enzyme. 

FIGURE 17.6 Allosteric effects in phosphofructokinase. At high [ATP], phosphofructokinase behaves cooperatively, and the plot of enzyme activity versus [fructose-6-phosphate] is sigmoidal. High [ATP] thus inhibits PFK, decreasing the enzyme s affinity for fructose-6-phosphate. 

The cooperative binding of Oj by hemoglobin, and the allosteric effects in many enzymes, requires interactions between sites that are widely separated in space. The present model was proposed in 1965 to... 

Regulatory or allosteric enzymes like enzyme 1 are, in some instances, regulated by activation. That is, whereas some effector molecules such as F exert negative effects on enzyme activity, other effectors show stimulatory, or positive, influences on activity. 

Ghanges in the availability of substrates are responsible for most changes in metabolism either directly or indirectly acting via changes in hormone secretion. Three mechanisms are responsible for regulating the activity of enzymes in carbohydrate metabolism (1) changes in the rate of enzyme synthesis, (2) covalent modification by reversible phosphorylation, and (3) allosteric effects. 

Some enzymes are controlled by both allosterism and covalent modification often brought about by hormone stimulation of the cell. Allosteric effects will take effect immediately because the enzyme is responding to local intracellular conditions of substrate or coenzyme concentrations, but covalent effects because they are driven by hormonal stimulation may take a little longer to have an impact but will be part of a coordinated response in several tissues of the body sensitive to the hormone. 

The key control enzyme in the pathway is glycogen synthase (GS) which occurs in either a high activity state (GS-a) or a low activity state (GS-b) the switch from one to the other is brought about partly by covalent modification of the enzyme in response to stimulation by glucagon and partly by allosteric effects of key metabolites. Glycogen... 

There are many examples of phosphorylation/dephosphorylation control of enzymes found in carbohydrate, fat and amino acid metabolism and most are ultimately under the control of a hormone induced second messenger usually, cytosolic cyclic AMP (cAMP). PDH is one of the relatively few mitochondrial enzymes to show covalent modification control, but PDH kinase and PDH phosphatase are controlled primarily by allosteric effects of NADH, acetyl-CoA and calcium ions rather than cAMP (see Table 6.6). 

Allosteric inhibitors bind to a separate binding site outside the active center (6). This results in a conformational change in the enzyme protein that indirectly reduces its activity (see p. 116). Allosteric effects practically only occur in oligomeric enzymes. The kinetics of this type of system can no longer be described using the simple Micha-elis-Menten model. 

Depending on the enzyme, allosteric effectors can influence the maximum rate V ax. the semi-saturation concentration [A]o.5, and the Hill coef dent h. If it is mainly V ax that is changed, the term V system is used. Much more common are K systems , in which allosteric effects only influence [A]o.5 and h. 

ID-myo-inositol 1,3,4,5-tetrakisphosphate <1> (<1> recombinant, catalytically active fragment of isoform C, allosteric product activation in the absence of Ca /calmodulin, which per se activates enzyme and abolishes the allosteric effect [31]) [31]... 

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