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Nuclear magnetic resonance studies of the catalytic mechanism of proteolytic enzymes. Ionization behavior of the histidine residue in the catalytic triad of alpha-lytic protease--implications for the catalytic mechanism of serine proteases. Ionization behavior of enzymic and inhibitor groups in the tetrahedral adduct between alpha-lytic protease and a peptide aldehyde. Kinetics of pepsin-catalyzed hydrolysis of N-tri-fluoroacetyl amino acids

Citation

Hunkapiller, Michael W. (1974) Nuclear magnetic resonance studies of the catalytic mechanism of proteolytic enzymes. Ionization behavior of the histidine residue in the catalytic triad of alpha-lytic protease--implications for the catalytic mechanism of serine proteases. Ionization behavior of enzymic and inhibitor groups in the tetrahedral adduct between alpha-lytic protease and a peptide aldehyde. Kinetics of pepsin-catalyzed hydrolysis of N-tri-fluoroacetyl amino acids. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-11072003-091804

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. PART I Selective [superscript 13]C enrichment of C-2 of the single histidine residue of the serine protease [alpha]-lytic protease has allowed direct study of the Asp-His-Ser catalytic triad by magnetic resonance techniques. Both the chemical shift of C-2 and the coupling between C-2 and its directly bonded hydrogen have been observed as a function of pH. The results indicate that only below pH 3.3 does the histidine imidazole ring become protonated and only above pH 6.7 does the aspartic acid residue lose a proton to generate a carboxylate anion. Over the pH range 3.3-6.7, the catalytic triad contains a neutral aspartic acid and neutral histidine residue--not the ionized forms hitherto assumed. This interpretation of the ionization characteristics of the catalytic triad leads to a proposed catalytic mechanism which avoids any requirement for unfavorable charge separation in the transition state. The histidine residue plays two roles: (i) it provides insulation between water and the buried carboxylate anion, thus ensuring the latter a hydrophobic environment, and (ii) it provides a relay for net proton transfer from serine hydroxyl to carboxylate anion. The aspartate anion acts as the ultimate base which holds a proton during catalysis. An anionic, rather than a neutral, base both avoids the necessity of charge separation and, by giving the catalytic locus an overall negative charge, assists preferential expulsion of product relative to substrate from the active site. Relaxation measurements (T[subscript 1], T[subscript 2], and nuclear Overhauser enhancement) indicate that, over the pH range of enzymic activity, the histidine residue is held rigidly within the protein. PART II Magnetic resonance techniques have been used to study ionization behavior of enzymic and inhibitor moieties in the tetrahedral adduct (hemiacetal) formed between [alpha]-lytic protease and a peptide aldehyde, N-Ac-L-Ala-L-Pro-L-alaninal. Chemical shift, coupling constant, and relaxation measurements of [superscript 13]C-enriched C-2 of the catalytic histidine residue indicate that at pH > 6.25 the complex contains neutral aspartic acid, neutral histidine, and negatively charged inhibitor. Below pH 6.25, both the inhibitor oxyanion and the histidine become protonated in a cooperative ionization process which forces the histidine from its rigidly-held position as a member of the catalytic triad into a solution-like environment. This behavior by a complex thought to resemble the transition state for serine protease-catalyzed hydrolysis of ester and amide substrates supports proposals for a catalytic mechanism which involves a minimum of charge separation in the transition state. It also attests to the power of the intricate hydrogen-bonding network (previously observed in x-ray diffraction studies) to stabilize an otherwise high-energy intermediate and thereby achieve catalysis. PART III The acidic gastric proteases, pepsin and gastricsin, have been found to catalyze hydrolysis of several N-trifluoroacetyl-L-amino acids with aromatic side chains. This catalytic activity is lost when they are chemically modified so as to inactivate their proteolytic activity. Magnetic resonance techniques were used to follow the porcine pepsin-catalyzed hydrolysis of N-trifluoroacetyl L-phenylalanine in the pH range 1.7-5.4. This study revealed that non-productive binding strongly influences the observed kinetic parameters and that productive enzyme-substrate binding requires an anionic substrate (pK[subscript a] 2.8) and an undissociated group (pK[subscript a] 3.7) on the free enzyme. Binding is also affected by ionization of a group on the free enzyme with a pK[subscript a] near 4.8. A kinetic isotope effect [...] has been observed for the reaction which suggests that proton transfer is involved in the rate-limiting step. A new mechanism--one involving three carboxylic acid groups on the enzyme and an intermediate in which the amino moiety is noncovalently held by the enzyme after release of the acyl moiety--is proposed to explain these and previous observations on catalysis by pepsin.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Awards:The Herbert Newby McCoy Award, 1974
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Richards, John H.
Thesis Committee:
  • Unknown, Unknown
Defense Date:10 April 1974
Record Number:CaltechETD:etd-11072003-091804
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-11072003-091804
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4434
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:10 Nov 2003
Last Modified:26 Dec 2012 03:08

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