Slow-binding inhibition

Slow, tight-binding inhibition occurs when slow-binding inhibition takes place at inhibitor concentrations comparable to that of the enzyme, in which case the previous two mechanisms can still apply. Comprehensive review articles on the subject of tight, slow, and slow, tight-binding inhibitors ate available in the literature (12,14). 

Palese P, Tobita K, Ueda M, Compans RW (1974b) Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61 397 10 Pegg MS, von Itzstein M (1994) Slow-binding inhibition of sialidase from influenza virus, Biochem Mol Biol Int 32 851-858... 

If the inhibition is found to be rapidly reversible, we must next determine if the approach to equilibrium for the enzyme-inhibitor complex is also rapid. As described in Chapter 4, some inhibitors bind slowly to their target enzymes, on a time scale that is long in comparision to the time scale of the reaction velocity measurement. The effect of such slow binding inhibition is to convert the linear progress curve seen in the absence of inhibitor to a curvilinear function (Figure 5.10). When nonlinear progress curves are observed in the presence of inhibitor, the analysis of... 

The hallmark of slow binding inhibition is that the degree of inhibition at a fixed concentration of compound will vary over time, as equilibrium is slowly established between the free and enzyme-bound forms of the compound. Often the establishment of enzyme-inhibitor equilibrium is manifested over the time course of the enzyme activity assay, and this leads to a curvature of the reaction progress curve over a time scale where the uninhibited reaction progress curve is linear. We saw... 

In this section we focus on differentiating slow binding inhibition due to the mechanisms shown in schemes B and C of Figure 6.3. 

On the other hand, when K Kf, the concentration of inhibitor required to observe slow binding inhibition would be much less than the value of K, for the inhibitor encounter complex. When, for example, the inhibitor concentration is limited, due to solubility or other factors, and therefore cannot be titrated above the value of Kif the steady state concentration of the El encounter complex will be kinet-ically insignificant. Under these conditions it can be shown (see Copeland, 2000) that Equation (6.6) reduces to... 

Scheme 1. The kinetic scheme for a) "classic" reversible inhibition, and b) slow-binding" inhibition.

Z Wu, CT Walsh. Phosphinate analogs of D-, D-dipeptides slow-binding inhibition... 

Z Wu, CT Walsh. Phosphinate analogs of D-,D-dipeptides slow-binding inhibition and proteolysis protection of VanX, a D-,D-dipeptidase required for vancomycin resistance in Enterococcus faecium. Proc Natl Acad Sci (USA) 92 11603-11607, 1995. 

Figure 13 Progress curve for slow-binding inhibition.

Transition-state inhibitors, especially those with peptidyl or peptidomimetic extensions, are slow-binding inhibitors, and the protease-inhibitor binding mechanism includes one or more weakly bound intermediates before the formation of the tightly bound E I complex. This slow-binding inhibition is a hallmark of inhibitors that bind in the active site in a substrate-like manner. In this way, transition-state analogs mimic the association... 

A good comparison of rapid reversible and slow-binding inhibition can be found in a recent study on the inhibition of arginase, an enzyme that catalyzes the hydrolysis of L-arginine to yield L-omithine and urea (Equation 17.31). 

It is difficult to find clear-cut examples of slow-binding inhibition occurring by mechanism A. However, the inhibition of Factor Xa by a peptidyl-a-ketothiazole was found to be unusual because it appeared that the formation of E. I was partially rate limiting. Factor is a trypsinlike protease found in the blood... 

Indicates that slow-binding inhibition was observed and that both the initial (larger number) and final values are reported. 

Methylumbelliferyl /V-acetylncuraminic acid, (MUNANA) (Sigma, St. Louis, MO). 2 mM stock solutions should be made up in either 10 mM CaCl2 and 100 mM sodium acetate buffer, pH 5.5 (IC50 determination) or water (slow-binding inhibition determination). 

Pegg MS, von Itzstein M. Slow-binding inhibition of sialidase from influenza virus. Bio-chem Mol Biol Int 1994 32 851-858. 

Most mechanisms for slow binding inhibition involve one or two kinetic steps during association of the inhibitor with the enzyme.32 In the one step mechanism, a high affinity complex (E-I, apparent inhibition constant K ), is generated directly, without any detectable intermediates. In the two-step process, there is an initial complex (E-I, apparent inhibition constant K ), in equilibrium with uninhibited enzyme, followed by a subsequent tightening to give the final complex (E-I ) with an overall steady state apparent inhibition constant K. ... 

Salowe, S. P., and Hermes, J. D. (1998). Competitive and slow-binding inhibition of calcineurin by drug x immunophilin complexes. Arch. Biochem. Biophys. 355, 165-174. 

W. Liu, C. J. Rogers, A. J. Fisher, M. D. Toney, Biochemistry 2002, 41, 12320-12328. Aminophosphonate inhibitors of dialkylglycine decarboxylase structural basis for slow binding inhibition. 

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