NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition

Journal Article · Tue Sep 11 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00488a028· OSTI ID:5706432

Weiss, M A ^[1]; Frank, B H; Heiney, R; Pekar, A ^[2]; Khait, I; Neuringer, L J ^[3]; Shoelson, S E ^[4]

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1. Harvard Medical School, Boston, MA (USA) Massachusetts General Hospital, Boston (USA)
2. Eli Lilly and Co., Indianapolis, IN (USA)
3. Massachusetts Institute of Technology, Cambridge (USA)
4. Harvard Medical School, Boston (USA)

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg{sup 31}-Arg{sup 32}) and a type II activity that cleaves the C-peptide/A-chain junction (Lys{sup 64}-Arg{sup 65}). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, the authors have undertaken comparative {sup 1}H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models or prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the {sup 1}H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substrates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic {sup 1}H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. They propose that this structure (designated the CA knuckle) provides a recognition element for type II proinsulin endopeptidase.

• OSTI ID: 5706432
• 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 ACID SEQUENCE
DICHROISM
ELECTROMAGNETIC RADIATION
ENZYMES
HORMONES
HYDROLASES
INSULIN
MAGNETIC CIRCULAR DICHROISM
MAGNETIC RESONANCE
MOLECULAR STRUCTURE
NEAR ULTRAVIOLET RADIATION
NONSPECIFIC PEPTIDASES
NUCLEAR MAGNETIC RESONANCE
PEPTIDE HORMONES
PEPTIDE HYDROLASES
POLARIZATION
RADIATIONS
RESONANCE
ULTRAVIOLET RADIATION