Proc. NatI. Acad. Sci. USA Vol. 89, pp. 3835-3839, May 1992 Biochemistry An unusual active site identified in a family of zinc metalloendopeptidases (insulin degrading enzyme/nitochondrial processing protease) ANDREW B. BECKER AND RICHARD A. ROTH Department of Pharmacology, Stanford University School of Medicine, Stanford, CA 94305 Communicated by Brian W. Matthews, January 13, 1992 (received for review December 6, 1991) ABSTRACT An unusual active site has been identified in dopeptidases, many of which show little overall sequence a family of zinc metalloendopeptidases that includes bacterial identity to thermolysin outside of this domain (4, 5). Re- protease M and the human and Drosophila insidn-degrading cently, through mutagenesis studies, the sequence HEXXH enzymes. All of these enzymes have been characterized as has been confirmed to be at the active site of several ofthese metalloendopeptidases and purified protease HI has been zinc-dependent proteases, including neutral endopeptidase shown to contain stoichiometric levels of zinc. However, all and angiotensin-converting enzyme (6-8). Moreover, this three proteases lack the consensus sequence (EIEXXH) de- consensus sequence has even been used to predict the scribed in the active site of other zinc metalloendopeptidases. peptidase activity ofproteins not previously considered to be Instead, these proteases contain an inversion of this motif, proteases, such as leukotriene A4 hydrolase (9). HXXEH. To determine whether this region could represent the However, some proteases, based on their amino acid active site in these proteins, the two histidines in protease m sequence, do not fit into the currently recognized classifica- were individually mutated to arginine and the glutamate was tion system. One example is the carboxypeptidases, zinc- mutated to glutamine. All three mutants were devoid of dependent metalloexopeptidases, in which the metal binding proteolytic activity toward an exogenous substrate, insulin, as domain consists of the motif HXXE(X)123H (4) as opposed to compared to the wild-type protease. Three lines of evidence the more common active site seen in thermolysin. Another indicate that this loss of activity in the mutants is not due to example is the bacterial enzyme protease III, or pi, from distortion of the three-dimensional structure of the protein: (i) Escherichia coli (10-13). This periplasmic enzyme shows the mutants are secreted into the periplasmic space and chro- 27% overall sequence identity with a human enzyme, called matograph normally; (ia) all three mutants are expressed at insulin-degrading enzyme (14), and 28% identity with a levels nearly identical to wild-type protein and do not appear homologous enzyme from Drosophila melanogaster (15). In to have an increased susceptibility to proteolysis in the bacteria; addition, three regions in these enzymes show >50% se- and (iii) the mutants compete equally with wild-type protein in quence identity (10, 14, 15). Although the physiological roles a radioimmunoassay. The purified wild-type and glutamate of these three enzymes are not known, they all degrade mutants were found to contain stoichiometric amounts of zinc insulin (10-17). All three proteases also have molecular by atomic absorption spectrophotometry, whereas both histi- masses of "100 kDa and, based on their requirement for dine mutants had negligible zinc signals. These findings are divalent cations, have been classified as metalloendopepti- consistent with this region being the active site in this protein, dases. Moreover, protease III has recently been shown to with the contain zinc at stoichiometric levels (18). However, none of histidine residues coordinating the essential zinc atom these enzymes contains the consensus active site sequence and the glutamate involved in catalysis. HEXXH described above for zinc metalloendopeptidases. A careful alignment of the sequences of these three pro- Proteases play a variety of roles in many physiological teases reveals the conservation of 12 histidines and gluta- processes, including fibrinolysis, blood homeostasis, com- mates, which could function in zinc binding (10, 14, 15). In plement activation, digestion, and hormone processing (1). fact, a number of motifs resembling that of the carboxypep- Based on common active sites and mechanisms of action as tidases can be identified in all three enzymes, including determined by primary amino acid sequence, inhibitor and HXXE(X)138H. In addition, all three enzymes contain a substrate profiles, and x-ray crystallographic analyses (2), sequence, HXXEH, which could be considered an inversion these proteins are generally classified into one of four fam- ofthe active site consensus sequence seen in thermolysin and ilies. Representative members of each protease class (i.e., other metalloendopeptidases (10, 14, 15). This sequence is cysteine, serine, aspartyl, and metallo-) have been crystal- found in one of the three highly conserved regions present in lized and their active sites elucidated (2). For example, the these enzymes. The surrounding residues of this sequence bacterial enzyme thermolysin is the prototypic zinc metal- also appear to be inverted in comparison to other zinc loendopeptidase. X-ray crystallographic studies have shown metalloendopeptidases (Table 1). The spacing of the two that this enzyme contains two histidines (His-142 and His- histidines and the first histidine to the glutamate in this region 146) and a glutamic acid (Glu-143) at its active site, with the are consistent with the one to three amino acids separating histidines coordinating the binding of a zinc atom and the the zinc binding residues in more than a dozen zinc enzymes glutamate acting as a general base in catalysis (3). Based on whose structures have been analyzed (4) and are the only two studies in thermolysin, it has been proposed that the active histidines and/or glutamates conserved among all three pro- site sequence in metalloendopeptidases is HEXXH (where X teases that exhibit the requisite spacing. is any amino acid, H is histidine, and E is glutamate). This To test whether the residues in the apparently inverted sequence has been identified in well over 15 zinc-dependent sequence HXXEH comprise the active site of protease III, metalloproteases, including aminopeptidases and metalloen- the two histidine residues were individually mutated to arginine and the glutamate was changed to glutamine by The publication costs ofthis article were defrayed in part by page charge site-directed mutagenesis. (The mutants were named by payment. This article must therefore be hereby marked "advertisement" using the single-letter amino acid code and indicating the in accordance with 18 U.S.C. §1734 solely to indicate this fact. number of the residue that was mutated-H88R, histidine-88 3835 Downloaded by guest on September 24, 2021 3836 Biochemistry: Becker and Roth Proc. Natl. Acad. Sci. USA 89 (1992) Table 1. Comparison of the proposed active site residues in Coomassie blue-stained gels, were pooled. The protease peak bacterial protease III, the human (hIDE) and Drosophila was concentrated to 1 ml in an Amicon chamber with a (dIDE) insulin-degrading enzymes, and several other YM-30 membrane (Amicon) and then applied to an Ultrogel zinc metalloendopeptidases AcA34 column (1.5 x 90 cm) equilibrated with a buffer Enzyme Sequence Residues Ref(s). containing 10 mM Hepes (pH 7.6) and 0.02% sodium azide. Again, the peak fractions as determined by Coomassie blue- . * ** Protease III GLA H YL ER MSL 85-95 10 stained gels were pooled and then concentrated in a Centri- hIDE GLS H FC EH MLF 106-116 14 con 30 (Amicon) to 1-1.5 ml. dIDE GLA H FC ER MLF 78-88 15 Insulin-Degradation Assays. The indicated amounts of pro- ** * . tein relative to wild-type protease III (determined by radio- Thermolysin VVA HE LT H AVT 139-149 3 immunoassays and Coomassie blue-stained gels) were incu- Collagenase VAA HE LG H SLG 215-225 19, 20 bated in microtiter wells previously coated with an antibody Serratia protease TFT HE IG H ALG 89-99 21 directed against protease III (18). The wells were washed and Endopeptidase 24.11 VIG HE IT H GFD 580-590 22,23 the immunocaptured enzyme was tested for its ability to Leukotriene A4 degrade 125I-labeled insulin (1251-insulin). Degradation of in- hydrolase VIA HE IS H SWT 292-302 9 sulin was assessed by its loss of ability to bind to the insulin The three active site residues, two histidines and a glutamate, in receptor as described (18). All assays were performed in a number of proteases are marked with an asterisk. Underlined triplicate using 40,000 cpm of 1251-insulin in a final vol of 35 residues indicate residues adjacent to the active site that are inverted p.l for 30 min at 370C. The assays were terminated by addition in the insulin-degrading enzymes and protease III relative to other of bacitracin to a final concentration of 1 mg/ml, and the metalloproteases. The diamond is used to mark residues that are extent of degradation was assessed in a receptor binding invariably hydrophobic in the different metalloendopeptidases. assay (18). Radioimmunoassay of Wild-Type and Mutant Protease III to arginine; H92R, histidine-92 to arginine; E91Q, gluta- Proteins. The indicated amounts of each protein were incu- mate-91 to glutamine.) The mutant proteins were then com- bated with 50,000 cpm of 251I-protease III on microtiter wells pared to wild-type protein in terms of activity and zinc previously coated with an antibody directed against protease content to ascertain whether this region is the active site in III (18). The binding was performed overnight at 40C and the this protein family. decrease in binding of iodinated protein to the antibody relative to a control with no competitor was calculated as the MATERIALS AND METHODS percent inhibition of binding. All assays were performed in triplicate. Materials. The following were purchased: standard SDS Metal Determinations. The amount of zinc in each purified molecular weight markers from Bio-Rad, bacitracin from protein preparation was determined by atomic absorption Sigma, phenyl-Sepharose from Pharmacia, Ultrogel AcA-34 spectrophotometry (Perkin-Elmer, model 2380).