Nonsticky substrates

The plot will have a slope of unity if Sr is very large but zero for a nonsticky substrate. In the general case, Sr is (slope)/(l - slope). 

The V/K profile is sensitive to the pK values of groups on the substrate or the enzyme form with which it combines that have a required protonation state for binding and/or catalysis. Since catalysis is usually more sensitive to correct protonation state than binding, one tends to see few partial effects in V/K profiles that is, when they begin to drop they keep on decreasing. For nonsticky substrates the profiles have simple shapes, and the pK values seen are the correct ones. Thus, if the physiological substrate is known to be sticky, one should always look for a slower alternate substrate to use for pH studies. 

Although the pH profiles for nonsticky substrates have simpler shapes and are easier to interpret, there is useful information in the pH profiles for a sticky substrate, and thus one must know what to expect, and how to interpret the results. The pK values may be perturbed in the V/K profile for a sticky substrate, and both the V/K and V profiles may show a hollow, or at least a flatter shape, near the pK. Consider the following mechanism in which protonation of a group with a pK less than 7 prevents catalysis ... 

With alanine dehydrogenase, (V/ Taianine) was 1.4 at the pH optimum and increased to 2.0 below pH 7, whereas (WATserine) was 2.0 at all pH values (serine is a slow, nonsticky substrate) (Si). The internal commitments appear to be the same for both substrates and are sizeable, since the intrinsic isotope effect is likely to be 5 or 6. In cases like this, a rough estimate of the stickiness of the substrate can be obtained from the following equation ... 

Substrates are normally sticky in the reaction direction with the higher maximum velocity, and not always then. In the direction with the lower maximum velocity, substrates cannot normally be sticky because their release rates from the enzyme must exceed the more rapid maximum velocity in the reverse direction. Thus, one needs to use the pH profiles of slow substrates only in the direction with the fastest maximum velocity in the reverse direction, one can use the profiles of the normal substrates for comparison. Further, one should use the V/K profiles for nonsticky substrates to determine the pK a values. Then, one should determine the nature of the catalytic groups by solvent perturbation and temperature variation of the pJta values, and then, by comparison with the V/K profile of the normal sticky substrate determine the stickiness and, from the shape of the profile in the vicinity of the pJCn, how rapidly the protonation state of the group is equilibrated when the substrate is present (Rose et al, 1974 Cleland, 1982). 

Obviously, the V profiles for the slow nonsticky substrate are the best choice, since these Vprofiles will show the correct piTa vcdues of amino acid side chains on enzyme in the enzyme-substrate complex that are responsible for catalysis. The apparent pK s of the catalytic groups of fumarase have been measured in both directions (Brandt et al, 1963). When malate is used as a substrate, Vprofile is beU-shaped showing two p a s, 6.4 and 9.0, respectively temperature variation of these groups indicates that p Ta 6.4 corresponds to dissociation of a carboxyl and pXa 9.0 to dissociation of a histidine side chain in the active site of enzyme. Thus, from the malate side of reaction, the enzyme is active if histidine is protonated and the carboxyl ionized. With fumarate as a substrate, the V profile is again bell-shaped showing two p a s. 7.0 and 4.9, respectively this time, however, the temperature variation indicates that p a 7.0 corresponds to dissociation of a carboxyl and p/iTa 4.9 to dissociation of a histidine side chain. Thus, from the fumarate side of reaction, the enzyme is active if carboxyl is protonated and histidine is not. 

The log (V7A versus pH profile for a nonsticky substrate shows the correct pKj, values of groups necessary for binding and catalysis. Those p Ta values not present in profiles are groups that act as acid-base catalysts during... 

This indicates a lack of dynamic cohesion within the adducts i.e. the substrate has considerable freedom for reorientation within the receptor. The apparent reason for an absence of mechanical coupling is the nearly cylindrical symmetry of cucurbituril, which allows the guest an axis of rotational freedom when held within the cavity. Hence, the bound substrates show only a moderate increase in tc relative to that exhibited in solution. No relationship exists between values and the thermodynamic stability of the complexes as gauged by K (or K, cf. Tables 1 and 2). It must be concluded that the interior of cucurbituril is notably nonsticky . This reinforces previous conclusions that the thermodynamic affinity within adducts is chiefly governed by hydrophobic interactions affecting the solvated hydrocarbon components, plus electrostatic ion-dipole attractions between the carbonyls of the receptor and the ammonium cation of the ligands. 

A fibrous substrate is impregnated with this mixture, heated for 5 min at 70 C and cooled to 22°C. The resultant impregnated material has slightly tacky faces. By passing the sheet between felt rollers saturated with an accelerator such as dimethylaniline, a smooth, nonsticky skin is formed on the sheet. 

The gecko adhesion system has extraordinary properties (1) is directional (2) attaches strongly with minimal preload (3) detaches quickly and easily (4) sticks to nearly every material (5) is self-cleaning (6) does not self-adhere and (7) is nonsticky by default (Autumn 2006). Gecko dry adhesion depends more on geometry than on chemistry of the substrate or the material of the fibrils - keratin proteins. 

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