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Angiotensin-converting enzyme 2 (ACE2), a homologue of ACE, represents a new and potentially important target in cardio-renal disease. A model of the active site of ACE2, based on the crystal structure of testicular ACE, has been developed and indicates that the catalytic mechanism of ACE2 resembles that of ACE. Structural differences exist between the active site of ACE (dipeptidyl carboxypeptidase) and ACE2 (carboxypeptidase) that are responsible for the differences in specificity. The main differences occur in the ligand-binding pockets, particularly at the S2' subsite and in the binding of the peptide carboxy-terminus. The model explains why the classical ACE inhibitor lisinopril is unable to bind to ACE2. On the basis of the ability of ACE2 to cleave a variety of biologically active peptides, a consensus sequence of Pro-X-Pro-hydrophobic/basic for the protease specificity of ACE2 has been defined that is supported by the ACE2 model. The dipeptide, Pro-Phe, completely inhibits ACE2 activity at 180 microM with angiotensin II as the substrate. As with ACE, the chloride dependence of ACE2 is substrate-specific such that the hydrolysis of angiotensin I and the synthetic peptide substrate, Mca-APK(Dnp), are activated in the presence of chloride ions, whereas the cleavage of angiotensin II is inhibited. The ACE2 model is also suggestive of a possible mechanism for chloride activation. The structural insights provided by these analyses for the differences in inhibition pattern and substrate specificity among ACE and its homologue ACE2 and for the chloride dependence of ACE/ACE2 activity are valuable in understanding the function and regulation of ACE2.

A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)