sup 15 N and sup 13 C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

Journal Article · Tue Sep 11 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00488a021· OSTI ID:5706434

Jaffe, E K; Rajagopalan, J S ^[1]; Markham, G D ^[2]

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1. Univ. of Pennsylvania School of Dental Medicine, Philadelphia (USA)
2. Fox Chase Cancer Center, Philadelphia, PA (USA)

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through {sup 13}C and {sup 15}N NMR. The authors knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by {sup 13}C or {sup 15}N NMR. Here they extend their {sup 13}C NMR studies to PBGS complexes with (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA and report {sup 15}N NMR studies of ({sup 15}N)ALA bound to PBGS. As in their previous {sup 13}C NMR studies, observation of enzyme-bound {sup 15}N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pK{sub a} is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent. For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C{sub 4} of ALA and an active-site lysine. The {sup 13}C chemical shift of (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between ({sup 15}N)ALA and hydrazine or hydroxylamine, the {sup 15}N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C{sub 4} of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.

• OSTI ID: 5706434
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:36; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMINO ACIDS
AMINOLEVULINIC ACID
BIOLOGICAL EFFECTS
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
CHEMICAL SHIFT
ENZYMES
ESTERS
EVEN-ODD NUCLEI
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
IMINES
ISOTOPES
LIGASES
LIGHT NUCLEI
MAGNETIC RESONANCE
METHYL METHANESULFONATE
MUTAGENS
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PORPHYRINS
REACTION INTERMEDIATES
RESONANCE
SCHIFF BASES
STABLE ISOTOPES
SUBSTRATES
SULFONIC ACID ESTERS