Enzymes, affinity binding

Chemical modifications like alkylation with (A-ethylmaleimide (NEM) or oxidation with diamide that inhibit the phosphorylation activity of the enzyme did not seem to have any significant effect on the high affinity binding site when the enzyme was solubilized in the detergent decyl-PEG [69,41]. However, in the intact membrane these treatments reduced the affinity by a factor of 2-3. The reduction of the affinity was exclusively due to modification of the cysteine residue at position 384 in the B domain [69]. Apparently, the detergent effects the interaction between the B and C domains. 

The interpretation of much of the binding data given so far is based upon the assumption that the high affinity binding sites represent a population of independent sites. In the unphosphorylated II" " these sites would open up either to the periplas-mic or cytoplasmic side of the membrane independently of each other. The assumption ignores the evidence that the enzyme is, in fact, multimeric and that the data... 

Figure 5.6 Biphasic concentration-response plot for an enzyme displaying a high- and low-affinity binding interaction with an inhibitor. In panel A, the data are fit to Equation (5.4) and the best fit suggests a Hill coefficient of about 0.46. In panel B, the data are fitted to an equation that accounts for two, nonidentical binding interactions Vj/v0 = (a/(l + ([/]/ICs0))) + ((1 - a)/(l+([t]/IC(o)))> where a is an amplitude term for the population with high binding affinity, reflected by IC , and IC 0 is the IC50 for the lower affinity interaction. (See Copeland, 2000, for further details.)...

Full and partial competitive inhibitory mechanisms, (a) Reaction scheme for full competitive inhibition indicates binding of substrate and inhibitor to a common site, (b) Lineweaver-Burk plot for full competitive inhibition reveals a common intercept with the 1/v axis and an increase in slope to infinity at infinitely high inhibitor concentrations. In this example, Ki = 3 pM. (c) Replot of Lineweaver-Burk slopes from (b) is linear, confirming a full inhibitory mechanism, (d) Reaction scheme for partial competitive inhibition indicates binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor affects the affinity of enzyme for substrate and the presence of substrate affects the affinity of enzyme for inhibitor, both by a factor a. (e) Lineweaver-Burk plot for partial competitive inhibition reveals a common intercept with the 1/v axis and an increase in slope to a finite value at infinitely high inhibitor concentrations. In this example, Ki = 3 pM and = 4. (f) Replot of Lineweaver-Burk slopes from (e) is hyperbolic, confirming a partial inhibitory mechanism... 

In partial (hyperbohc) mixed inhibition (O Figure 4-12d), binding of inhibitor to a site distinct from the active site results in altered affinity of enzyme for substrate (by a factor, ot) as well as a change (by a factor, /i) in the rate at which product can be released from ESI. The effects of a partial mixed inhibitor on a Lineweaver-Burk plot depend upon the actual values, and on the relative values, of ot and fl. Once again, inhibitor plots can intersect the control plot above or below, but not on, the oeaxis, and to the left or to the right of, but not on, the y-axis. Because Vmax cannot be driven to zero, a maximum Lineweaver-Burk slope is reached at infinitely high inhibitor concentrations beyond which no further increase occurs. 

The reduction in enzymatic activity that results from the formation of nonproductive enzyme complexes at high substrate concentration. The most straightforward explanation for substrate inhibition is that a second set of lower affinity binding sites exists for a substrate, and occupancy of these sites ties up the enzyme in nonproductive or catalytically inefficient forms. Other explanations include (a) the removal of an essential active site metal ion or other cofactor from the enzyme by high concentrations of substrate, (b) an excess of unchelated substrate (such as ATP" , relative to the metal ion-substrate complex (such as CaATP or MgATP ) which is the true substrate and (c) the binding of a second molecule of substrate at a subsite of the normally occupied substrate binding pocket, such that neither substrate molecule can attain the catalytically active conformation". For multisubstrate enzymes, nonproductive dead-end complexes can also result in substrate inhibition in the presence of one of the reaction... 

One alternative method for the determination of enzyme activities which is particularly effective at low enzyme concentrations involves enrichment with the enzyme by affinity binding (preferably of the reversible type) to an affinity column in the enzyme thermistor unit. The enzyme activity is determined by introducing a pulse of excess substrate. 

---