NUCLEAR MAGNETIC RESONANCE STUDIES OF SERINE PROTEINASES (NMR)

BRIAN TOBIAS, Purdue University

Abstract

Nuclear magnetic resonance (NMR) spectroscopy has been applied to the study of serine proteinases at subzero temperature, and to the investigation of the exchange kinetics of the C$\sp{\varepsilon}$ and N$\sp{\delta}$ protons of His-57 of serine proteinases. The viscosities of cryosolvents commonly used for enzymology at subzero temperatures increases as the temperature is lowered. In order to probe the limits that this viscosity imposes on $\sp{13}$C NMR studies of enzymes, longitudinal and transverse NMR relaxation measurements were made with a $\sp{13}$C probe in the active site of $\alpha$-chymotrypsin. Transverse and longitudinal relaxation measurements of (1-$\sp{13}$C, 90%) acetyl-chymotrypsin indicated that the acyl-chymotrypsin was in the $\omega\tau\sb{\rm c}$ $>>$ 1 region where T$\sb 1$ is proportional to $\tau\sb{\rm c}$ and T$\sb 2$ is proportional to 1/$\tau\sb{\rm c}$. Since the viscosity of organic/water mixtures typically used for cryoenzymology dramatically increases as the temperature is lowered, the correlation time of the protein will increase. This sharply limits the temperature range in which enzyme-bound species can be studied by $\sp{13}$C NMR. Bovine pancreatic trypsin inhibitor (BPTI) forms extremely stable complexes with a number of serine proteinases. The crystal structure of the complex between BPTI and bovine trypsin (R. Huber and W. Bode (1978) Accounts of Chemical Research 11, 114-122) indicates that His-57 is shielded from solvent. It was found nevertheless that the exchange rate of the N$\sp{\delta}$ proton of His-57, which is hydrogen bonded to Asp-102, is up to 10$\sp9$ times faster than the half-time for dissociation of the complex. The original assignments (J. L. Markely and I. B. Ibanez (1978) Biochemistry 17, 4627-4646) of $\sp1$H NMR peaks to the C-2 protons of the two histidine residues of chymotrypsinogen A (His-40 and His-57), but not the assignments in $\alpha$-chymotrypsin, have been questioned recently (W. Schultz, Ph.D. Thesis, 1984). Since it was found that the C-2 proton of one histidine in chymotrypsinogen A exchanges with deuterium from $\sp2$H$\sb2$O solvent much more rapidly than the other, it was possible to use the differentially-exchanged protein to correlate histidine peak assignments in the zymogen and enzyme. After complete exchange of the C-2 proton of His-40, the residual signal due to the C-2 proton of His-57 was followed upon activation of chymotrypsinogen A to $\alpha$-chymotrypsin. The results firmly support the original assignments of Markley and Ibanez.

Degree

Ph.D.

Subject Area

Biochemistry

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