Studies on the Catalytic Mechanism of a Serine Protease from Streptomyces griseus

Abstract

The kinetics of the Streptomyces griseus protease-3-catalyzed hydrolysis of p-nitrophenylacetate have been studied by stopped-flow techniques with widely varied initial concentrations of enzyme and substrate. The results obtained are consistent with a three-step mechanism in which there is a rapid equilibration between enzyme and substrate to form a Michaelis complex with a dissociation constant in the order of 10 mM, followed by a moderately rapid formation of an acyl-enzyme and a rate-limiting deacylation of this intermediate. Estimated rate constants for the latter two reaction steps are 15 s-l and 0.09 s-l, respectively.The presence of a nucleophile such as methanol results in a partitioning of the deacylation process, reflected by a linear dependence of the maximum steady-state reaction velocity on the concentration of methanol. This observation provides confirmatory evidence for a rate-limiting deacylation step in the catalytic mechanism.Pronase, the extracellular proteolytic enzyme preparation from Streptomyces griseus, contains several components with protease activity [l, 21, some of which have been shown to be serine proteases [3-51. One of these serine enzymes (herein referred to as Streptomyces griseus protease 3) has received particular attention since it, despite its microbiological origin, exhibits striking sequential similarities to pancreatic serine enzymes [6] and has a broad substrate specificity showing similarities to both chymotrypsin and elastase [4,7].Acyltransfer reactions to water or other nucleophilic acceptors, catalyzed by serine or thiol hydrolases, have often been interpreted in view of the minimal kinetic scheme s P1

Effect of Boric Acid on the Catalytic Activity of Streptomyces griseus Protease 3

“…The typical oscilloscope traces shown in Fig.3 illustrate that boric acid affects the rate of the transient burst of p-nitrophenol formation obseived on mixing Streptomyces griseus protease 3 with p-nitrophenylacetate a t pH 7.5 [9], and that identical traces ale obtained in the presence of boric acid irrespectively of the choice of pre-mixing conditions (substrate and inhibitor mixed with enzyme, or substrate mixed with enzyme pre-equilibrated with inhibitor). The latter observation strongly indicates that boric acid equilibrates rapidly with the enzyme, under which condition scheme (i) predicts that the apparent first-order rate-constant ktr for the transient burst is determined by Fig.…”

“…Previous investigations have revealed several striking sequential [ 1111 and mechanistical [ 1,9] similarities between Streptomyces griseus protease 3 and chymotrypsin. The data reported here, in fact, suggest that the two proteins, despite their wide phylogenetic and physiological differences, may be considered as homologous, a t least as concerns the structure and function of the catalytic site around the reactive seryl residue Further studies of Streptomyces griseus protease 3 will now be directed mainly towards the substrate-specificity of the protein, which appears to be considerably broader than that of chymotrypsin [2].…”

“…1 shows that boric acid acts as a competitive inhibitor of the enzymatic hydrolysis of p-nitrophenylacetate a t pH 7.5. Since deacylation is ratelimiting with this ester substrate (k, < k, [1,9]) the k,-term in Eqn (2) may be complet,ely neglected, and the observation that kcat remains unaffected by boric acid thus provides evidence that binding of the inhibitor to the acyl-enzyme is kinetically insignificant ( K , = 0). The estimate of K4 obtained on fitting Eqn (3) to the data in Fig.…”

Foams Made of Engineered Recombinant Spider Silk Proteins as 3D Scaffolds for Cell Growth

“…Although PXIV is more likely a suitable model for digestive enzymes, it was here used as an additional model enzyme to allow comparison with previously published data. 48,49 In correspondence to previous degradation experiments of other eADF4- C16) foams with different salt-to-protein ratios (low ratio: 75:1 (4%), 50:1 (6%), and 37.5:1 (8%); high ratio: 125:1 (4%), 85:1 (6%), and 60:1 (8%) (w/w) salt/protein ratio) and salt particle size ranges as indicated (I, 63− 125; II, 125−315; III, 315−500). (B) Compressive moduli of 8% foams were significantly higher in comparison to 4% and 6% foams at low salt-toprotein ratios (75:1 (4%), 50:1 (6%), and 37.5:1 (8%)) (**p ≤ 0.001, ***p < 0.0001).…”

“…The protease mix XIV is a mixture of at least ten proteases with broad substrate specificity. Although PXIV is more likely a suitable model for digestive enzymes, it was here used as an additional model enzyme to allow comparison with previously published data. , In correspondence to previous degradation experiments of other eADF4­(C16) morphologies (particles, films and nonwoven meshes) with PXIV and CHC the total amount of 87.5 μg protease per 1 mg scaffold was kept constant, resulting in a concentration of 465 μg/mL, which is a factor of 1300 higher than the naturally occurring 351 ng/mL of wound proteases . Degradation experiments were not feasible at lower protease concentrations, due to the slow kinetics and to the extremely low concentration of degradation products (being below the detection limit).…”