Phenylalanine-35, which is a residue of the hydrophobic patch on the surface of cytochrome b5, has been mutated into Tyr35, His35 and Leu35 to elucidate the functions of the Phe35 and give further insight into the roles of the hydrophobic patch and/or aromatic network. The effects of these mutations on the heme environment, denaturation towards heating and the denaturant urea, redox potential and stability of protein were studied. The relative stability of cytochrome b5 and its mutants towards heating has the order Phe35Tyr > wild type > Phe35Leu > Phe35His in the oxidized state and wild type > Phe35Tyr > Phe35Leu > Phe35His in the reduced state. All the mutants exhibit decreased reduction potentials: Phe35Tyr -66 mV, Phe35His -51 mV and Phe35Leu -28 mV, which are more negative than that of the wild type. The order of redox potential reflects the relative stability in the oxidized and reduced states. A method of producing multiple mutants at a single site of a gene is also described for the first time.

The major function of the regulatory (R) subunit ofthe cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R-C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RH) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RH and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RH in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RU and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RU could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RUI cAMP was increased by addition ofthe C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RH subunit may interact with the C subunit in a cAMP-dependent manner.The cAMP-dependent protein kinase (PKA) consists of regulatory (R) and catalytic (C) subunits that associate to form an inactive tetrameric holoenzyme (R2C2). The binding of cAMP to the R subunit leads to dissociation of the inactive holoenzyme and the release of the active C subunit (1).Two major types of PKA have been classified based on the isozymic identity of the R subunit, which exists in two forms, RI and RII (2,3). Both forms of the R subunit share the general domain structure, which has been defined by limited proteolysis and affinity labeling (4). The 50 residues at the amino terminus of the R subunit are essential for dimerization to R2 (5). Two tandem cAMP-binding domains are located at the carboxyl terminus of the R subunit (6, 7). The sequence of these cAMP binding domains is homologous to that of the catabolite gene activator protein (CAP) from Escherichia coli, and a p-barrel structural model of cAMP-binding sites has been proposed, based on the crystallographic coordinates of CAP (8-10). The two cAMP binding sites (sites A and B) of the R subunit have different preferences for cAMP analogs and display different dissociation rates for cAMP (11,12).A proteolytically sensitive "hinge" region (residues 90-100, see Fig. 1) has been emphasized thus far as the site of interaction with the C subunit. The hinge region in RII contains a substrate consensus sequence, Arg-Arg-Xaa-Ser, while that in RI has a pseudosubstrate sequence,...