Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.

Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase

Inhibition of insulin-regulated aminopeptidase (IRAP) has been demonstrated to facilitate memory in rodents, making IRAP a potential target for the development of cognitive enhancing therapies. In this study, we generated a 3-D model of the catalytic domain of IRAP based on the crystal structure of leukotriene A4 hydrolase (LTA4H). This model identified two key residues at the ‘entrance’ of the catalytic cleft of IRAP, Ala427 and Leu483, which present a more open arrangement of the S1 subsite compared with LTA4H. These residues may define the size and 3-D structure of the catalytic pocket, thereby conferring substrate and inhibitor specificity. Alteration of the S1 subsite by the mutation A427Y in IRAP markedly increased the rate of substrate cleavage V of the enzyme for a synthetic substrate, although a corresponding increase in the rate of cleavage of peptide substrates Leu-enkephalin and vasopressin was was not apparent. In contrast, [L483F]IRAP demonstrated a 30-fold decrease in activity due to changes in both substrate affinity and rate of substrate cleavage. [L483F]IRAP, although capable of efficiently cleaving the N-terminal cysteine from vasopressin, was unable to cleave the tyrosine residue from either Leu-enkephalin or Cyt6-desCys1-vasopressin (2–9), both substrates of IRAP. An 11-fold reduction in the affinity of the peptide inhibitor norleucine1-angiotensin IV was observed, whereas the affinity of angiotensin IV remained unaltered. In additionm we predict that the peptide inhibitors bind to the catalytic site, with the NH2-terminal P1 residue occupying the catalytic cleft (S1 subsite) in a manner similar to that proposed for peptide substrates.

Identification and characterization of a new class of cognitive enhancers based on inhibition of insulin-regulated aminopeptidase

Approximately one-quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment, underscoring the need for effective cognitive-enhancing agents. Insulin-regulated aminopeptidase (IRAP) is potentially an innovative target for the development of cognitive enhancers, as its peptide inhibitors exhibit memory-enhancing effects in both normal and memory-impaired rodents. Using a homology model of the catalytic domain of IRAP and virtual screening, we have identified a class of nonpeptide, small-molecule inhibitors of IRAP. Structure-based computational development of an initial "hit" resulted in the identification of two divergent families of compounds. Subsequent medicinal chemistry performed on the highest affinity compound produced inhibitors with nanomolar affinities (Ki 20–700 nM) for IRAP. In vivo efficacy of one of these inhibitors was demonstrated in rats with an acute dose (1 nmol in 1 µl) administered into the lateral ventricles, improving performance in both spatial working and recognition memory paradigms. We have identified a family of specific IRAP inhibitors that is biologically active which will be useful both in understanding the physiological role of IRAP and potentially in the development of clinically useful cognitive enhancers. Notably, this study also provides unequivocal proof of principal that inhibition of IRAP results in memory enhancement.

Membrane bound members of the M1 family : more than aminopeptidases

In mammals the M1 aminopeptidase family consists of nine different proteins, five of which are integral membrane proteins. The aminopeptidases are defined by two motifs in the catalytic domain; a zinc binding motif HEXXH-(X18)-E and an exopeptidase motif GXMEN. Aminopeptidases of this family are able to cleave a broad range of peptides down to only to a single peptide. This ability to either generate or degrade active peptide hormones is the focus of this review. In addition to their capacity to degrade a range of peptides a number of these aminopeptidases have novel functions that impact on cell signalling and will be discussed.

Arg1098 is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity

Angiotensin (Ang) I-converting enzyme (ACE) is a Zn²+ metalloprotease with two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl dipeptidases. Hydrolysis by ACE of its decapeptide substrate Ang I is increased by Cl−, but the molecular mechanism of this regulation is unclear. A search for single substitutions to Gln among all conserved basic residues (Lys/Arg) in human ACE C-domain identified R1098Q as the sole mutant that lacked Cl− dependence. Cl−dependence is also lost when the equivalent Arg in the N-domain, Arg⁵⁰⁰, is substituted with Gln. The Arg¹⁰⁹⁸ to Lys substitution reduced Cl− binding affinity by ∼100-fold. In the absence of Cl−, substrate binding affinity (1/K m) of and catalytic efficiency (k cat/K m) for Ang I hydrolysis are increased 6.9- and 32-fold, respectively, by the Arg¹⁰⁹⁸ to Gln substitution, and are similar (<2-fold difference) to the respective wild-type C-domain catalytic constants in the presence of optimal [Cl−]. The Arg¹⁰⁹⁸ to Gln substitution also eliminates Cl− dependence for hydrolysis of tetrapeptide substrates, but activity toward these substrates is similar to that of the wild-type C-domain in the absence of Cl−. These findings indicate that: 1) Arg¹⁰⁹⁸ is a critical residue of the C-domain Cl−-binding site and 2) a basic side chain is necessary for Cl− dependence. For tetrapeptide substrates, the inability of R1098Q to recreate the high affinity state generated by the Cl−-C-domain interaction suggests that substrate interactions with the enzyme-bound Cl− are much more important for the hydrolysis of short substrates than for Ang I. Since Cl− concentrations are saturating under physiological conditions and Arg¹⁰⁹⁸ is not critical for Ang I hydrolysis, we speculate that the evolutionary pressure for the maintenance of the Cl−-binding site is its ability to allow cleavage of short cognate peptide substrates at high catalytic efficiencies.

The last 10 amino acid residues beyond the hydrophobic motif are critical for the catalytic competence and function of protein kinase Cá*

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKCα is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ~60% of the catalytic activity of the mutant PKCα, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKCα in immune complex kinase assays. The PKCα C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKCα immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKCα immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKCα is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKCα function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

The very C-terminus of protein kinase CĹ is critical for the full catalytic competence but its hydrophobic motif is dispensable for the interaction with 3-phosphoinositide-dependent kinase-1

In this article, we explore the role of the C-terminus (V5 domain) of PKCvar epsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCvar epsilon resulted in a PKCvar epsilon-Δ731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCvar epsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCvar epsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCvar epsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCvar epsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.

Angiotensin I-converting enzyme transition state stabilization by HIS1089 : evidence for a catalytic mechanism distinct from other gluzincin metalloproteinases

Angiotensin (Ang) I-converting enzyme (ACE) is a member of the gluzincin family of zinc metalloproteinases that contains two homologous catalytic domains. Both the N- and C-terminal domains are peptidyl-dipeptidases that catalyze Ang II formation and bradykinin degradation. Multiple sequence alignment was used to predict His¹⁰⁸⁹ as the catalytic residue in human ACE C-domain that, by analogy with the prototypical gluzincin, thermolysin, stabilizes the scissile carbonyl bond through a hydrogen bond during transition state binding. Site-directed mutagenesis was used to change His¹⁰⁸⁹ to Ala or Leu. At pH 7.5, with Ang I as substrate, k_cat/K_m values for these Ala and Leu mutants were 430 and 4,000-fold lower, respectively, compared with wild-type enzyme and were mainly due to a decrease in catalytic rate (k_cat) with minor effects on ground state substrate binding (K_m). A 120,000-fold decrease in the binding of lisinopril, a proposed transition state mimic, was also observed with the His¹⁰⁸⁹ --> Ala mutation. ACE C-domain-dependent cleavage of AcAFAA showed a pH optimum of 8.2. H1089A has a pH optimum of 5.5 with no pH dependence of its catalytic activity in the range 6.5-10.5, indicating that the His¹⁰⁸⁹ side chain allows ACE to function as an alkaline peptidyl-dipeptidase. Since transition state mutants of other gluzincins show pH optima shifts toward the alkaline, this effect of His¹⁰⁸⁹ on the ACE pH optimum and its ability to influence transition state binding of the sulfhydryl inhibitor captopril indicate that the catalytic mechanism of ACE is distinct from that of other gluzincins.