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Six analogues of glutathione disulfide were synthesized. All of them involved the abolishment of charges, either by amidation of carboxylates or by removal of amino groups. Four of these analogues could be bound to crystalline oxidized glutathione reductase, and their binding modes could be established by X-ray analyses at 2.4-A resolution. All six analogues were catalytically processed; the kinetic parameters were determined. The two analogues that did not bind in the crystals had by far the poorest catalytic efficiencies. Kinetic parameters together with X-ray data show the influence of each charged group on binding and catalytic rate. Data analysis indicates that the enzyme avoids processing of incorrect substrates in two ways: First, it reduces their binding strengths and/or enforces displacement of catalytically important substrate parts. Furthermore, it forms a fragile cluster of bound substrate and catalytically competent residues, which is unbalanced by incorrect parts of the substrate such that catalysis is prevented. A scouting microcalorimetric study using glutathione disulfide yielded a binding enthalpy of -103 (+/- 10) kJ/mol at 25 degrees C and a heat capacity change of -8 (+/- 1) kJ.mol-1.K-1. The study showed that it is feasible to measure these parameters as a function of substrate modification.

The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP P MgADP + ADP) from 1. Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were 2. Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by 3. Thc N-terminus is blocked by an acetyl group as shown by proton magnetic resonance. 4. Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2-iodosobenzoic acid cleavages.5. The enzyme is a monomer of 220 amino acids with M , 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putativc activesite region of the enzyme. After position 11 1, however, there is an insertion of 32 residues in thc yeast species, similar to the adenylate kinase and the GTP : AMP phosphotransferase from beef heart mitochondria.baker's yeast has been determined. sequenced with either a solid-phase sequencer or a gas-phase sequencer. [5] and of two isozymes from beef heart mitochondria [6, 7] have been reported. An X-ray diffraction analysis of porcine adenylate kinase at 0.3 nm in conjunction with the sequence yielded the spatial structure [8]. Whereas the active-site region of the enzyme could be assigned unambiguously by localization of substrate analogues at 0.6-nm resolution 191, the exact binding positions of the substrates are still not known. However, the observed binding sites were challenged by nuclear magnetic resonance analyses [lo]. This controversy is likely to be resolved by study of adenylate kinase from yeast (AKy), because it cocrystallizes with the potent enzyme inhibitor Ap,A as well as with the natural substrate MgATP (Noda and Tomasselli, unpublished results). These crystals are presently being analyzed by X-ray diffraction. This paper presents the complete amino acid sequence of AKy, which together with the forthcoming X-ray analysis should yield the exact substrate binding geometry, leading us a step closer to the elucidation of the reaction mechanism. MATERIALS AND METHODS MaterialsBaker's yeast (Red Star Yeast, Universal Foods Corporation, Milwaukee, WI) was kindly donated by the manufacturer. Clostripain from Clostridiurn histolyticurn, endoproteinase Lys-C from Lysohacter enzyrnogenes, pronase from Streptornyces griseus (Boehringer Mannheim), Staphylococcus aureus V 8 protease (Miles Laboratories Inc.) and trypsin pretreated with L-3-tosyl-amido-2-phenylethylchloromethylanc (Worthington) were used for proteolytic cleavages. Cyanogen bromide (Fluka) and 2-iodosobenzoic acid (Serva) were employed for chemical cleavages. Reagents and solvents for the gas-phase sequencer were from Applied Biosystems. All other chemicals were of analytical grade. Protein preparation and chemical mod(ficationsAKy, prepared and assayed as in [l 11, had a specific activity of 1833 U/mg and was homogeneous by sodium dodecyl sulfate/pol...

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