Volume 138, number 2 FEBS LETTERS February 1982 PROTON NMR SPECTROSCOPY OF THE ACTIVE SITE HISTIDINE OF a-LYTIC PROTEINASE Effects of adjacent 13C and lSN labels William M. WESTLER, John L. MARKLEY and William W. BACHOVCHIN* Department of Chemistry, Purdue University, West Lafayette, IN 47907 and *Department of Biochemistry and Pharmacology, Tufts University, School of Medicine, Boston, iliA 02111, USA Received 23 December 1981 I. Introduction A histidine auxotroph of Lysobacter enzymogenes (ATC 29847) was grown on media containing either isotopically labeled [90% 13CqL- or [90% lSN~, 90% ~SNe]D,L-histidine. The enzyme, a-lytic proteinase (EC 3.4.21.12), was isolated from these cultures as well as from cultures of wild-type bacteria grown on unlabeled medium. ~H NMR spectra at 360 MHz obtained with all 3 purified enzymes revealed that the only important differences concerned a resonance previously assigned to the Ce-H proton of the active site histidine [1 ]. a-Lytic proteinase has only a single histidine residue [2] located at position 57 in the chymotrypsinogen numbering scheme [2,3 ]. Presence of the adjacent lSN labels broadened the histidine Ce-H peak by about a factor of 2 by unresolved scalar coupling. Presence of a directly bonded ~3C led to dis- appearance of the histidine Ce-H peak by a combina- tion of scalar coupling and dipolar broadening. These effects should be useful for the cross-assignment of IH NMR peaks of 13C and lSN enriched proteins. The 13C and lSN labeled proteins were found to undergo the reversible a-b conformational transition [4] which changes the pK' a of His s7 from 6.5-5.9. 2. Experimental procedures The Lysobacter enzymogenes culture was a gift from Dr F. D. Cook. Preparation of the histidine auxotroph was as in [5]. Cultures were grown, and t~.lytic proteinase was isolated by minor modification [5] of the procedures in [6]. The [lSNS,15Ne]HisSLa- lytic proteinase used was that in [5]. 90% Isotopically labeled [13Ce]L-histidine was purchased from KOR Isotopes (Cambridge MA). The purity and degree of enrichment were verified by IH NMR and 13C NMR spectroscopy. 360 MH_z1H NMR spectra were obtained with a Nicolet NT-360 spectrometer located in the Purdue University Biochemical Magnetic Resonance Laboratory. Usual procedures were used in handling the NMR samples [7]. Chemical shifts are reported in ppm from internal sodium 2,2-dimethyl- 2-sflapentane-5-sulfonate (DSS). a-Lytic proteinase can exist in two conformational forms: (i) that obtained by dialysis against water and then lyophili- zation and which is stable at low pH (conformer a); (ii) that arising after incubation of conformer a for ~14 h at neutral pH (conformer b) [4,8]. The b-con- former of the enzyme can be reconverted to the a-con- former by dialysis and lyophilization. The 2 forms are not the result of dimerization, and both forms are catalytically active [4]. All 3 enzymes studied (13C- or ~SN-labeled and unlabeled) exhibited the conforma- tional transition which can be followed by ~ H-NMR. Spectra of the a-conformer are reported here; how- ever, the b-conformer gives His Ce-H spectra very similar to those shown. The a-b transition results in extensive changes in the ~H NMR spectrum of the enzyme; the change can be monitored most conve- niently by following the intensity of a methionine methyl peak which shifts from 2.09 ppm in the a-form to 2.12 ppm in the b-form [4]. 3. Results and discussion The aliphatic and aromatic regions of the 360 MHz Published by Elsevier Biomedical Press 00145793/82/0000-0000/$02.75 © 1982 Federation of European Biochemical Societies 233