Abstract
The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Our results suptionship between thiol ester cleavage and protein in- port a model in which thiol ester cleavage permits a activation, the rateofs methylamine incorporation and conformational change in a2M that entraps and sterithiol release were measured
The inactivation followed the same time course as a conformational change in a2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limitedproteolysis.,themethyla2-Macroglobulinis a n M, = 725,000 plasma protease inhibitorcomposed of four identical subunits(1,Z)
The first is the site of limited proteolysis by bound proteases and ilsocated near the centeorf the subunit peptide chain [1].The second functional region is located about 200 amineinactivation of a2M is a sequentialtwo-step amino residues away from the first and is a reactive group process where thiol ester cleavage is followedby a with the propertiesof a thiol ester[4,5].Namely, eachsubunit proteinconformationalchange
Summary
MM ( B ) ,75 mM (c)a,nd 100 mM (D). of the differences in reported rates and reaction conditions. A plot of the terminal slope uersus methylamine concentration is shownin Fig. 4.The results shown in Fig. 4 suggest that there aretwo methylamine-dependent steps, the thiol ester cleavage and the conformational change. In fully methylamine-modified a2M,
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