The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.

The effect of cellulase size on hydrolysis was studied by comparing the behavior of crosslinked cellulase (CC) with normal cellulase (FC). The average molecular weight of the CC was at least three times the molecular weight of the FC. The amounts of each enzyme were adjusted so that the degree of solubilization after 2 h was the same. The degree of solubilization of Avicel with CC was higher than that with FC in the late stage of reaction. The degree of solubilization of pretreated lignocelluloses was much greater than that of Avicel, but the degree of solubilization with CC was lower than that with FC at all times during the reaction. The degree of solubilization of artificial lignified Avicel was higher with FC than with CC, but the degree of solubilization of de-lignified the artificial lignified Avicel was lower with FC than with CC. The degree of solubilization of amorphous cellulose with FC was the same as that with CC at all times during the reaction. These behaviors are examined by the hypothesis that when small pores dominate, the smaller enzyme components diffuse into the pores and become inactive since synergism with the larger components is no longer possible, whereas, when larger pores dominate, the entire enzyme can diffuse in and therefore the available surface area is increased. This hypothesis is supported by direct measurement of the pore size in two of the substrates and by diffusion inside Avicel of only smaller molecular cellulase component.

Reduction in the activity and the concentration of the adsorbed enzyme are noted in the experimental data. Two alternative mechanisms, inactivation of the adsorbed enzyme and mass transfer of the enzyme from the bulk solution to the solution within the cellulose fibril where the cellulase is assumed to be inactive, are used to represent the decline in activity. The decline in concentration of the adsorbed enzyme is represented by a modest product inhibition and, more importantly, the assumption that the concentration of the adsorption sites is proportional to the square of the remaining substrate concentration. Measurements of both adsorbed enzyme and product concentration over time are used in determining parameter values. The model is applied to a series of experiments having a 10-fold range of substrate concentration and to an experiment in which the product is removed continuously. For both deactivation mechanisms, a very good representation of product concentration (standard deviation 3.6%) is obtained over the full period (168 h) of hydrolysis; the representation of adsorbed enzyme is, however, less accurate (standard deviation 6.7-6.8%).