Andrew C. Storer

Structure of rat procathepsin B: model for inhibition of cysteine protease activity by the proregion

BACKGROUND Cysteine proteases of the papain superfamily are synthesized as inactive precursors with a 60-110 residue N-terminal prosegment. The propeptides are potent inhibitors of their parent proteases. Although the proregion binding mode has been elucidated for all other protease classes, that of the cysteine proteases remained elusive. RESULTS We report the three-dimensional structure of rat procathepsin B, determined at 2.8 A resolution. The 62-residue proregion does not form a globular structure on its own, but folds along the surface of mature cathepsin B. The N-terminal part of the proregion packs against a surface loop, with Trp24p (p indicating the proregion) playing a pivotal role in these interactions. Inhibition occurs by blocking access to the active site: part of the proregion enters the substrate-binding cleft in a similar manner to a natural substrate, but in a reverse orientation. CONCLUSIONS The structure of procathepsin B provides the first insight into the mode of interaction between a mature cysteine protease from the papain superfamily and its prosegment. Maturation results in only one loop of cathepsin B changing conformation significantly, replacing contacts lost by removal of the prosegment. Contrary to many other proproteases, no rearrangement of the N terminus occurs following activation. Binding of the prosegment involves interaction with regions of the enzyme remote from the substrate-binding cleft and suggests a novel strategy for inhibitor design. The region of the prosegment where the activating cleavage occurs makes little contact with the enzyme, leading to speculation on the activation mechanism.

Detection of covalent enzyme‐substrate complexes of nitrilase by ion‐spray mass spectroscopy

Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of M r 40 258 ±2 and 40 388 ±2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher M r, species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate‐limiting.

Recent insights into cysteine protease specificity : Lessons for drug design

Cysteine proteases of the papain superfamily are usually considered to possess a relatively broad substrate specificity. However, despite a number of similarities between cysteine proteases, unique and/or restricted preferences for substrates have been served in a few cases and differences do exist that can form the basis for the design of more specific inhibitors. Recent crystallographic measurements coupled to mutational analysis have allowed the molecular basis behind several of these specificity determinants to be uncovered.

Nature of papain products resulting from inactivation by a peptidyl O-acyl hydroxamate.

Mass spectrometry has been used to provide insights into the mechanism of inhibition of cysteine proteases by a hydroxylamine derivative, CBZ‐Phe‐Gly‐NH‐O‐CO‐(2,4,6‐Me3)Ph. An oxidized form of papain resulting from the incubation of the enzyme with the peptidyl hydroxamate in the absence of a reducing agent has been identified as a sulfinic acid. The presence of a covalent enzyme‐inhibitor complex of molecular mass consistent with a sulfenamide adduct of papain could also be detected by this method. Implications on the mechanism of inactivation of cysteine proteases by peptidyl hydroxamates are discussed.

Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity.

Synthetic peptides derived from the proregion of rat cathepsin B were used to identify functionally important regions and residues for cathepsin B inhibition. Successive 5 amino acid deletions of a 56 amino acid propeptide from both the N‐ and C‐termini has allowed the identification of two regions important for inhibitory activity: the NTTWQ (residues 21p–25p) and CGTVL (42p–46p) regions. Alanine scanning of residues within these two regions indicates that Trp‐24p and Cys‐42p contribute strongly to inhibition, their replacement by Ala resulting in 160‐ and 140‐fold increases in K i , respectively.

SYNTHESIS OF AMIDRAZONES USING AN ENGINEERED PAPAIN NITRILE HYDRATASE

To demonstrate the usefulness of an engineered papain nitrile hydratase as a biocatalyst, a peptide amidrazone was prepared by incubation of the nitrile MeOCO‐Phe‐Ala‐nitrile with the Gln19Glu papain mutant in the presence of salicylic hydrazide as a nucleophile. The amidrazone results from nucleophilic attack by salicylic hydrazide at the imino carbon of the thioimidate adduct formed between the enzyme and the peptide nitrile substrate. Compared to wild‐type enzyme, the engineered nitrile hydratase causes a better than 4000‐fold increase in the rate of amidrazone formation and yields a product of much higher purity. The advantages over other nitrile‐hydrolyzing enzymes and current limitations of the papain nitrile hydratase are discussed.

The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B

Human cathepsin B, the most abundant lysosomal cysteine protease, has been implicated in a variety of important physiological and pathological processes. It has been known for a long time that like other lysosomal cysteine proteases, cathepsin B becomes inactivated and undergoes irreversible denaturation at neutral or alkaline pH. However, the mechanism of this denaturation process remains mostly unknown up to this day. In the present work, nuclear magnetic resonance spectroscopy was used to characterize the molecular origin of the neutral‐pH inactivation and the refolding barrier of human cathepsin B. Two forms of human cathepsin B, the native form with Cys‐29 at the active site and a mutant with Cys‐29 replaced by Ala, were shown to have well‐folded structures at the active and slightly acidic condition of pH 5. Surprisingly, while the native cathepsin B irreversibly unfolds at pH 7.5, the C29A mutant was found to maintain a stable three‐dimensional structure at neutral pH conditions. In addition, replacement of Cys‐29 by Ala renders the process of the urea denaturation of human cathepsin B completely reversible, in contrast to the opposite behavior of the wild‐type cathepsin B. These results are very surprising in that replacement of one single residue, the active‐site Cys‐29, can eliminate the neutral‐pH denaturation and the refolding barrier. We speculate that this finding may have important implications in understanding the process of pH‐triggered inactivation commonly observed for most lysosomal cysteine proteases.

An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide

Selected fragments of the 62‐residue proregion (or residues 1p–62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p–51p and 26p–51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p–51p fragment, but remained in the 26p–43p and 28p–43p peptide fragments. Residues 23p–26p or TWQ25A in the N‐terminal 1p–29p fragment did not show cathepsin B‐induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p–51p peptide. Both the 1p–29p and 36p–51p fragments lack a common set of hydrophobic residues 30p–35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p–51p, 26p–51p, 28p–43p and 26p–43p, but the same residues induce the aggregation of peptides 16p–36p and 1p–36p. The peptide fragment 26p–43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p–43p or 26p–43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full‐length cathepsin B proregion.

Engineering of proteases and protease inhibition.

Proteases are unquestionably the single most studied class of enzymes and yet many questions still remain about their mechanisms and roles. Protein engineering offers the opportunity to provide some of the answers. In this review, recent advances towards the understanding of stability, mechanism, specificity and regulation of proteases and their inhibitors are outlined. In addition, the application of this increased understanding is also discussed.

Vibrational spectra and rotational isomerism of simple dialkyl dithioesters and N-acylglycine ethyl dithioesters, thiolesters, and dioxygenesters

Abstract Simple dialkyl dithioesters and N-acylglycine ethyl dithioesters have been investigated by Raman, resonance Raman and infrared spectroscopies. Based on the data of isotopic substituted derivatives and a normal coordinate analysis, vibrational assignments have been proposed for methyl dithioacetate. Raman spectra of ethyl dithioacetate(CH 3 C(=S)S CH 2 CH 3 ) and methyl dithiopropionate (CH 3 CH 2 C (=S)SCH 3 ) have provided fundamental knowledge on rotational isomerism about the bonds indicated. Raman, resonance Raman and FTIR spectroscopic studies on N-acylglycine ethyl dithioesters indicated the coexistence of three conformers in solution and a combined x-ray crystallographic-resonance Raman spectroscopic approach has been used to set up precise structure-spectra correlations for these conformers. The corresponding conformational states of N-acylglycine thiolesters and dioxygenesters are also discussed briefly.