THE ROLE OF ENZYME LYSYL AMINO GROUPS IN THE REACTION WITH α₂‐MACROGLOBULIN^a

Abstract

The primary observation, from our laboratory and others, of the effect of blocking the lysyl amino groups of enzymes is the reduction in the fraction of complexes that are resistant to SDS. The blocked enzyme derivatives do cause the specific proteolysis of the alpha 2M subunit to the 85K/100K fragments, and do cause the appearance of new thiol groups. With respect to the sequence of reaction, we may summarize the results by saying that if the reversible DMM-trypsin is, in fact, a model for the native enzyme, proteolysis can precede formation of the presumed covalent bond between bound enzyme and inhibitor. If our preliminary observations are borne out by later experiments, thiol release may precede covalent bond formation or loss of reactivity with amines, suggesting that an intact thiolester need not be the immediate target for amines; another intermediate, possibly the internal pyroglutamate originally proposed by Howard et al. and seen in model studies, may be an additional, or even the primary, target for covalent bonding with native enzymes. With regard to the "trap" hypothesis, the limited release of thiols in a slow phase is suggestive of enzyme activity within the alpha 2M-protease complex, consistent with the theory. Noncovalent irreversible complexes, however, are not a necessary part of associations seen with lysyl-blocked enzymes (which do cause proteolysis and do release thiols); this result is supported by limited data with noncovalently bound native enzymes. Some fraction of irreversible noncovalently bound enzymes may occur, but our results suggest that although alpha 2M-bound enzymes are unusually sterically hindered, the transformation to the presumed covalent state that appears to depend on intact amino groups, may be sufficient to explain the low dissociation of native enzymes. We feel that more experimental evidence is needed to resolve some of the ambiguities on this question but, we feel the existence of a "trapping" reaction has not been proved. In fact, given the possible existence of equilibria between covalent and noncovalent complexes observed, for example, in soybean trypsin inhibitor, and the very low dissociation constants observed with traditional protein-protein complexes, the question of physically encapsulated structures in alpha 2M may not be resolvable without direct evidence from crystal structures.

The time course of resolution of adhesions during fibrinolytic therapy in tetracycline-induced pleural injury in rabbits

“…Molecular-cage-type (51) complexes between ␣M and endogenous or exogenous uPA have previously been found in both airway fluids and PFs from humans and rabbits (35,37,38). Whereas proteinases complexed with ␣Ms interact with LMW molecules (substrates, inhibitors, ligands), they are sterically protected from highmolecular-weight (HMW) ligands (13,55). Their activities toward HMW substrates and inhibitors, if they exist, are greatly suppressed (35,62).…”

Kinetics of the reaction of thrombin and α2-macroglobulin

“…The final material was eluted with 0.1 M-EDTA, pH 7.0, dialysed against 20 mM-sodium citrate buffer, pH 7.0, and stored in 2 ml portions at -70 'C. The activity of a2M was assayed by protection of inhibition of esterase activity by soya-bean trypsin inhibitor, active-site titration and thiol-group release (Howard et al, 1980;Wu et al, 1981;Feinman et al, 1983). Proteins and reagents Human a-thrombin was a gift from Dr. John W. Fenton, II, of the New York State Department of Health, Albany, NY, U.S.A. 4,4'-Dithiodipyridine was obtained from Sigma Chemical Co. and was used without further purification.…”

Limited Mutations in Full-length Tetrameric Human α2-Macroglobulin Abrogate Binding of Platelet-derived Growth Factor-BB and Transforming Growth Factor-β1

“…For this reason, conformationally transformed ␣ 2 M is referred to as "activated." ␣ 2 M thiol ester bonds, which are formed by the side chains of Cys 949 and Glu 952 , also become exposed during ␣ 2 M conformational change and may react with lysine residues of the attacking protease to form covalent linkages; however, these linkages are not necessary to stabilize the ␣ 2 M-protease complex because the trapping mechanism is essentially irreversible under physiological conditions (7,8). ␣ 2 M conformational change may be induced in the absence of proteases by aminolysis of the thiol ester bonds with small primary amines such as methylamine (MA) (9,10).…”