Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.

Abstract

Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of I'4CIATP or 114CIADP in a manner that depends linearly on the extent of inactivation by either NN'-p-phenylenedimaleimide or Co(II)phenanthroline/ICo(IIIXphenanthroline)2CO31+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 -5 days). The nucleotide incorporated is not covalently bound because HC104 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the 'y-phosphate of ly-32PJATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-#,B,y-imidoltriphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with NN'-p-phenylenedimaleimide or cobalt phenanthroline complexes. There is a large body of information available indicating that the myosin molecule and its active proteolytic subfragments, double-headed heavy meromyosin and single-headed subfragment 1 (SF-1), undergo spectroscopically sensitive conformational changes during ATP binding, hydrolysis, and product release (refs. 1-5; see ref. 6 for review). Binding of MgADP results in a well-documented (7-10) increase in the reactivity of one of two critical SH groups, so-called SH-2, suggesting this thiol moves from a buried to an exposed position.Reisler and coworkers (11) have reported that these critical thiols, SH-1 and SH-2, can be crosslinked by a 12-to 14-A thiol crosslinking reagent, N,N'-p-phenylenedimaleimide (pPDM). Furthermore, Burke and Reisler (12) reported that crosslinking with shorter bifunctional thiol reagents occurred optimally when the crosslinking was performed in the presence of MgADP. This suggested these SH groups could move from 14 A to as close as 7 A upon binding of MgADP. We have recently provided further support for the proximal distance between SH-1 and SH-2 of 3-5 A based on evidence of simultaneous chelation of these two SH groups by a single-exchange-inert Co(III) phenanthroline complex (13,14).Inactivations with pPDM (11) or cobalt phenanthroline complexes (13) are greatly stimulated by the addition of MgADP. Surprisingly, as shown here, adenosine 5-[t,-yimidoltriphosphate (AdoPP[NHIP) does not bind to SF-1 inactivated in the presence of MgADP with either reagent. These apparently conflicting observations are explained by the finding that nucleotide is trapped at the active site during nucleotidestimulated inactivation. We provide evidence that the nucleotide is specifically trapped at the hydrolytic site and is only slowly released over a period of days. The trapped nucleotide is rapidly released when the enzyme deriva...

Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.

“…The finding that SH-1 becomes crosslinkable to either the 25-kDa or the 50-kDa region is consistent with the earlier reports that a spin label attached to SH-1 is more mobile in the presence of nucleotide (22,23) and that the crosslinking between SH-1 and SH-2 is accelerated by the presence of nucleotide (5,6). The observation that Mg-AdoPP[NH]P has an effect similar to that of Mg-ATP shows that an active ATPase cycle is not necessary to make the 50-kDa region crosslinkable to SH-1.…”

Dynamic interaction between cardiac myosin isoforms modifies velocity of actomyosin sliding in vitro.

“…Treatment of myosin with pPDM in the presence of Mg-ADP results in cross-linking of the two critical thiols of subfragment 1 (SH1 and SH2) and in concomitant trapping of Mg-ADP at the active site. [36][37][38] According to Chalovich et al,39 pPDM-treated myosin binds to actin with an affinity similar to myosin * ATP and myosin * ADP. Pi which are categorized as weakly bound states in the kinetic scheme of actomyosinATPase.40 Furthermore, pPDM-treated myosin remains in this state without splitting ATP (noncycling).…”

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

“…We conclude that the two possible sulfhydryl groups that react with the two heads of these reagents are sufficiently close together to react with the longer crosslinking reagents. As was observed in previous work using these reagents (16,26) Examination of the deduced primary structure of the maize enzyme (22) reveals that, of the seven total cysteinyl residues per monomer, two are at the putative dinucleotide-binding site at positions 192 (adjacent to the proposed NADP-binding site) and 246 (adjacent to the NAD-binding site). The amino acid sequences that bind NAD(P) are highly conserved among different enzymes that use these cofactors.…”

“…On the other hand, the presence of two sulfhydryl groups on NADP-malic enzyme that confers a different reactivity toward the bifunctional reagent cannot be ruled out. These maleimides were previously studied as crosslinkers of the y subunit of the chloroplast-coupling factor (16,25) and of the myosin subfragment I (26).…”