A new structural class of serine protease inhibitors revealed by the structure of the hirustasin-kallikrein complex.

BACKGROUND: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. RESULTS: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. CONCLUSIONS: The crystal structure of the kallikrein-hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel beta sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

Phosphorylation and inactivation of yeast fructose-1,6-bisphosphatase by cyclic AMP-dependent protein kinase from yeast.

Purified fructose-1,6-bisphosphatase from Saccharomyces cerevisiae was phosphorylated in vitro by purified yeast cAMP-dependent protein kinase. Maximal phosphorylation was accompanied by an inactivation of the enzyme by about 60%. In vitro phosphorylation caused changes in the kinetic properties of fructose-1,6-bisphosphatase: 1) the ratio R(Mg2+/Mn2+) of the enzyme activities measured at 10 mM Mg2+ and 2 mM Mn2+, respectively, decreased from 2.6 to 1.2; 2) the ratio R(pH 7/9) of the activities measured at pH 7.0 and pH 9.0, respectively, decreased from 0.62 to 0.38, indicating a shift of the pH optimum to the alkaline range. However, the affinity of the enzyme for its inhibitors fructose-2,6-bisphosphate (Fru-2,6-P2) and AMP, expressed as the concentration required for 50% inhibition, was not changed. The maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase was 0.6-0.75 mol/mol of the 40-kDa subunit. Serine was identified as the phosphate-labeled amino acid. The initial rate of in vitro phosphorylation of fructose-1,6-bisphosphatase, obtained with a maximally cAMP-activated protein kinase, increased when Fru-2,6-P2 and AMP, both potent inhibitors of the enzyme, were added. As Fru-2,6-P2 and AMP did not affect the phosphorylation of histone by cAMP-dependent protein kinase, the inhibitors must bind to fructose-1,6-bisphosphatase in such a way that the enzyme becomes a better substrate for phosphorylation. Nevertheless, Fru-2,6-P2 and AMP did not increase the maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase beyond that observed in the presence of cAMP alone.

Cyclic AMP and fructose-2,6-bisphosphate stimulated in vitro phosphorylation of yeast fructose-1,6-bisphosphatase.

Phosphorylation of purified yeast fructose-1,6-bisphosphatase was studied using purified preparations from yeast of two different cyclic AMP-independent protein kinases and a cyclic AMP-dependent protein kinase. Incorporation of 32P into fructose-1,6-bisphosphatase could be demonstrated only with the cyclic AMP-dependent protein kinase. Phosphorylation of fructose-1,6-bisphosphatase was stimulated by 3 microM fructose-2,6-bisphosphate and inhibited by 1 mM 5'-AMP.

Characterization of phosphorylase kinase activities in yeast.

Two phosphorylase kinase activities were resolved by DEAE-cellulose chromatography. The main activity peak was enriched 2800-fold, the minor appeared to be an aggregate of the enzyme. Phosphorylase kinase also phosphorylated histone and casein with no changes in phosphorylation ratios throughout the preparation steps but was most active on yeast phosphorylase. The molecular weight was 29000 +/- 2000. ATP, UTP, GTP served as substrates while CTP was inactive. Mg-ions activated the kinase without inhibition at high concentrations (30 mM). In addition to this cAMP-independent kinase, cAMP-dependent protein kinase also phosphorylated phosphorylase. The catalytic subunit and phosphorylase kinase were not identical since the latter was not inhibited by yeast cAMP binding protein.