Functional interaction between amino-acid residues 242 and 290 in cytochromes P-450 2B1 and 2B11

Abstract

Previous studies have revealed the functional importance of the negatively charged amino-acid residue Asp-290 of the phenobarbital-inducible dog liver cytochrome P-450 (P-450) 2B11 (Harlow, G.R. and Halpert J.R. (1996) Arch. Biochem. Biophys. 326, 85–92). A search for P-450 2B11 residues capable of forming a charge pair with Asp-290 suggested the positively charged residue Lys-242 as a likely candidate. Replacement of Lys-242 with Asp in a P-450 2B11 fusion protein with rat NADPH-cytochrome P-450 reductase (reductase) resulted in very low holoenzyme expression levels in Escherichia coli, as did replacement of Asp-290 with Lys. Remarkably, however, expression levels of the double mutant Lys-242→Asp/Asp-290→Lys were dramatically increased above either single replacement alone. Similarly, the pair-wise substitutions Lys-242→Leu/Asp-290→Ile in P-450 2B11 and Leu-242→Lys/Ile-290→Asp in P-450 2B1 showed greater holoenzyme expression levels than the constituent single mutants, providing further evidence for the close proximity of these residues within the three-dimensional structure of these two enzymes. These results support the hypothesis that a functional interaction exists between residues 242 and 290, which may help to coordinate the relative positions of proposed helices G and I. All of the mutant combinations, including the additional P-450 2B11 double mutants Tyr-242/Asn-290 and Tyr-242/Ser-290, displayed altered stereoselectivity of androstenedione hydroxylation.

Introduction

The cytochrome P-450 superfamily comprises a large group of hemoproteins that are capable of metabolizing a wide variety of both endogenous and exogenous compounds. Recent work in this laboratory has primarily focused on the structural basis of substrate specificity of various enzymes of the P-450 2B subfamily, including rat 2B1 1, 2, 3, 4, 5, 6, 7, 8, rabbit 2B4 and 2B5 9, 10, 11, and the phenobarbital-inducible dog liver P-450 2B11 10, 12. In previous studies, the replacement of Asp-290 in P-450 2B11 with Ile, which is the equivalent residue in rat P-450 2B1, altered the metabolism of androstenedione (AD), testosterone, 7-ethoxycoumarin, (R)- and (S)-warfarin, and 2,2′,4,4′,5,5′-hexachlorobiphenyl [12]. The lack of charge on these substrates suggested that Asp-290 may have a structural rather than direct catalytic role. It was subsequently shown that both the charge and size of residue 290 in P-450 2B11 have functional significance [13]. In that study, all eleven substitutions tested for Asp-290 in P-450 2B11 resulted in decreased enzyme activities. The replacement D290E yielded the highest activity (55% of wild-type), while substituting the positively charged amino-acid Arg for Asp-290 created an enzyme with the lowest activity (<1% of wild-type activity).

In the absence of an X-ray crystal structure for any mammalian P-450, computer-aided modeling of P-450 2B1 7, 8based on analogy with bacterial enzymes of known crystal structure, suggests that residue 290 of P-450 2B1 may be relatively buried inside the enzyme. The energetic cost of inserting a charged residue into a low dielectric environment is high, but inserting a salt bridge into a hydrophobic environment can be energetically much more favorable [14]. Mutagenesis studies of salt bridges buried within hydrophobic regions of other proteins have demonstrated that these types of interactions play a crucial role in determining protein conformation or folding dynamics 15, 16, 17, 18, 19. However, the exact role of buried salt bridges is not certain and is a subject of current interest [20].

Residue-residue interactions can be very valuable in confirming enzyme structure predictions. Recent results with P-450 2C enzymes have demonstrated that certain residues in positions predicted by alignment with bacterial P-450s interact with each other [21], thus validating a portion of the proposed P-450 2C three-dimensional structure. In the present study, heterologous expression of site-specifically mutated forms of dog P-450 2B11 and rat P-450 2B1 were used to study whether residue 242 forms a functional interaction with residue 290. Our analysis identifies a potential salt bridge between Asp-290 and Lys-242 in P-450 2B11, which apparently helps determine protein folding, protein stability, and/or enzyme active-site characteristics.