The calcium adenosinetriphosphatase of sarcoplasmic reticulum, preincubated with Ca2+ on the vesicle exterior (cE X Ca2), reacts with 0.3-0.5 mM Mg X ATP to form covalent phosphoenzyme (E approximately P X Ca2) with an observed rate constant of 220 s-1 (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4, 23 microM free external Ca2+, intact SR vesicles passively loaded with 20 mM Ca2+). If the phosphoryl-transfer step were rate-limiting, with kf = 220 s-1, the approach to equilibrium in the presence of ADP, to give 50% EP and kf = kr, would follow kobsd = kf + kr = 440 s-1. The reaction of cE X Ca2 with 0.8-1.2 mM ATP plus 0.25 mM ADP proceeds to 50% completion with kobsd = 270 s-1. This result shows that phosphoryl transfer from bound ATP to the enzyme is not the rate-limiting step for phosphoenzyme formation from cE X Ca2. The result is consistent with a rate-limiting conformational change of the cE X Ca2 X ATP intermediate followed by rapid (greater than or equal to 1000 s-1) phosphoryl transfer. Calcium dissociates from cE X Ca2 X ATP with kobsd = 80 s-1 and ATP dissociates with kobsd = 120 s-1 when cE X Ca2 X ATP is formed by the addition of ATP to cE X Ca2. However, when E X Ca2 X ATP is formed in the reverse direction, from the reaction of E approximately P X Ca2 and ADP, Ca2+ dissociates with kobsd = 45 s-1 and ATP dissociates with kobsd = 35 s-1. This shows that different E X Ca2 X ATP intermediates are generated in the forward and reverse directions, which are interconverted by a conformational change.

The kinetics for dissociation of the stable enzyme-calcium complex of the sarcoplasmic reticulum calcium ATPase, cE.Ca2, were followed by assay with simultaneous addition of [32P]ATP and EGTA, which gives 70% phosphorylation of cE.Ca2 with k = 300 s-1 (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). The binding of ATP to cE.Ca2 is described by kATP = 1.0 X 10(7) M-1 s-1, k-ATP = 120 s-1, and Kdiss = 12 microM; ATP binding is partially rate limiting for phosphorylation at less than 100 microM ATP. The sequential dissociation of Ca2+ from cE.Ca2 is described by k-2 = 55-60 s-1 for the first, "outer" Ca2+, k-1 = 25-30 s-1 for the second, "inner" Ca2+, and K0.5 = 3.4 microM, n = 1.9 (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). Dissociation of the inner Ca2+ is inhibited by external Ca2+, with K0.5 = k-1/k2 = 0.7 microM. This confirms the conclusion that dissociation of the two Ca2+ ions is sequential. The ability of cE.Ca2 to catalyze phosphorylation by ATP disappears in the presence of EGTA with k = 50-55 s-1, the same as k-2 for dissociation of the outer Ca2+ ion. This result, and the absence of the induction period that would occur if both cE.Ca2 and cE.Ca1 were catalytically competent, shows that both Ca2+ ions are required for phosphorylation. This conclusion is confirmed by the stoichiometry of 1.4/0.7 = 2.0 for the ratio of Ca2+ internalized to phosphoenzyme formed after simultaneous addition of ATP and EGTA. Phosphorylation of cE.Ca2 in the presence of 45Ca gives 0.15, not 0.3, 45Ca internalized, which corresponds to exchange of only 1 Ca2+ and is in agreement with this conclusion. The requirements for binding of two Ca2+ for catalytic specificity toward ATP and loss of two Ca2+ from E approximately P.Ca2 for specificity toward water account for the stoichiometry of Ca2+ transport and provide a possible reason for the two steps in the phosphorylation of cE.Ca2.ATP.

The binding of Ca2+ and the resulting change in catalytic specificity that allows phosphorylation of the calcium ATPase of sarcoplasmic reticulum by ATP were examined by measuring the amount of phosphoenzyme formation from [32P]ATP, or 45Ca incorporation into vesicles, after the simultaneous addition of ATP and EGTA at different times after mixing enzyme and Ca2+ (25 degrees C, pH 7.0, 5 mM MgSO4, 0.1 M KCl). A "burst" of calcium binding in the presence of high [Ca2+] gives approximately 12% phosphorylation and internalization of two Ca2+ at very short times after the addition of Ca2+ with this assay. This shows that calcium binding sites are available on the cytoplasmic-facing side of the free enzyme. Calcium binding to these sites induces the formation of cE.Ca2, the stable high-affinity form of the enzyme, with k = 40 s-1 at saturating [Ca2+] and a half-maximal rate at approximately 20 microM Ca2+ (from Kdiss = 7.4 X 10(-7) M for Ca.EGTA). The formation of cE.Ca2 through a "high-affinity" pathway can be described by the scheme E 1 in equilibrium cE.Ca1 2 in equilibrium cE.Ca2, with k1 = 3 X 10(6) M-1 s-1, k2 = 4.3 X 10(7) M-1 s-1, k-1 = 30 s-1, k-2 = 60 s-1, K1 = 9 X 10(-6) M, and K2 = 1.4 X 10(-6) M. The approach to equilibrium from E and 3.2 microM Ca2+ follows kobsd = kf + kr = 18 s-1 and gives kf = kr = 9 s-1. The rate of exchange of 45Ca into the inner position of cE.Ca2 shows an induction period and is not faster than the approach to equilibrium starting with E and 45Ca. The dissociation of 45Ca from the inner position of cE.45Ca.Ca in the presence of 3.2 microM Ca2+ occurs with a rate constant of 7 s-1. These results are inconsistent with a slow conformational change of free E to give cE, followed by rapid binding-dissociation of Ca2+.

The preferred amino acid residues at the P;and P2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P' or P2positions, where a set of 15 or 16 amino acids was introduced.Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termin, which allowed direct sequence analysis by We report here a rapid method for the quantitative determination of endoproteinase substrate specificity for subsites on the C-terminal (P')** side of the scissile bond of a peptide substrate that largely circumvents these limitations. This method'uses the proteinase-catalyzed competitive hydrolysis of defined peptide mixtures to map the subsite preferences of the proteinase. The' substrate is a mixture of N-acetylated peptides, identical in sequence except for a single position. The strategy is outlined in the example shown in Fig. 1 The exact determinants of 3C proteinase specificity are not well understood. In poliovirus, each of the eight 3C-specific polyprotein cleavage sites occurs between glutamine-glycine bonds; however, not all glutamine-glycine pairs in the polyprotein are cleaved, suggesting additional sequence or conformational determinants. In' other picornaviruses, the known or predicted polyprotein cleavage sites are not limited to glutamine-glycine pairs; the P' residues are replaced in some picornaviral polyproteins by serine, alanine, methionine, threonine, or valine (3,4

A new high-yield yeast expression/secretion system has been adapted for the plant thiol endoprotease papain. The propapain gene, obtained from Carica papaya fruit, is expressed in the yeast Saccharomyces cerevisiae. The gene was cloned into a FLAG epitope-tagging expression vector downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence. Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2). Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture supernatant where it accumulates to approximately 1.7 mg/l. The proregion contains three consensus N-linked glycosylation sites, whereas there are only two such sites in previously reported cDNA sequences. Removal of this third N-linked glycosylation site results in a drastic reduction in the level of protease activity present in the culture supernatant. Two different types of affinity chromatography were used to purify either propapain or papain. The propapain precursor is autoproteolytically activated to mature papain (M(r) = 24 kDa) using conditions reported previously. The kinetic parameters obtained agree well with the literature values. The yields of active papain are 10-fold higher than those previously reported for propapain in other yeast or bacterial expression systems. This, together with the ease with which mutant proteins can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.

No abstract

Mutations in the Escherichia coli promoter PBAD have been constructed which alter the spacing of the adjacent RNA polymerase and araC inducer protein binding sites. While deletion of a single base-pair or small insertions do not detectably affect araC protein binding to DNA and they do not alter the conserved sequence of the RNA polymerase binding site, stimulation of PBAD in vivo is greatly reduced. The experiments suggest that the distance or angle between the two proteins on the DNA is critical for promoter function. INTRODUCTIONTranscription from the Escherichia coli L-arabinose operon promoter,

The controversial question as to whether the ketoreductase activity of the animal fatty acid synthase is lost on dissociation of the homodimer has been addressed by using immobilized subunits which cannot reassociate under the conditions of assay. Ketoreductase activity, assessed with the model substrate S-acetoacetyl-N-acetylcysteamine, was identical in immobilized monomers and dimers, exhibiting normal Michaelis-Menten kinetics with Km values in the millimolar range. When acetoacetyl-CoA was used as a substrate, however, biphasic kinetics were observed in the case of the dimer, with estimated Km values in the micro- and milli-molar ranges, but only the high-Km reaction was observed with the monomer. Thus when the ketoreductase activities of the monomer and dimer are assessed with acetoacetyl-CoA at concentrations sufficient to saturate only the low-Km reaction, it appears that the ketoreductase activity towards acetoacetyl-CoA is lost upon dissociation. Reduction of acetoacetyl-CoA via the low-Km pathway is CoA-dependent, indicating that acetoacetyl-CoA can react with the dimer by two mechanisms: a high-Km pathway analogous to that utilized by model substrates and a low-Km pathway in which substrate and product are transferred between acyl-CoA and acyl-enzyme forms. The results indicate that the ketoreductase activity per se is unaffected by subunit dissociation and are consistent with a model in which the transfer of substrate from CoA ester to the acyl-carrier-protein domain necessitates juxtaposition of the transferase active-site serine residue of one subunit and the phosphopantetheine moiety of the adjacent subunit.