Plasticity of extended subsites facilitates divergent substrate recognition by Kex2 and furin

Laura Rozan; Damian J Krysan; Nathan C Rockwell; Robert S Fuller

doi:10.1074/jbc.M405362200

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Plasticity of extended subsites facilitates divergent substrate recognition by Kex2 and furin

Journal article

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Plasticity of extended subsites facilitates divergent substrate recognition by Kex2 and furin

Laura Rozan, Damian J Krysan, Nathan C Rockwell and Robert S Fuller

The Journal of biological chemistry, Vol.279(34), pp.35656-35663

08/20/2004

DOI: 10.1074/jbc.M405362200

PMID: 15159396

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Abstract

Yeast Kex2 and human furin are subtilisin-related proprotein convertases that function in the late secretory pathway and exhibit similar though distinguishable patterns of substrate recognition. Although both enzymes prefer Arg at P(1) and basic residues at P(2), the two differ in recognition of P(4) and P(6) residues. To probe P(4) and P(6) recognition by Kex2p, furin-like substitutions were made in the putative S(4) and S(6) subsites of Kex2. T252D and Q283E mutations were introduced to increase the preference for Arg at P(4) and P(6), respectively. Glu(255) was replaced with Ile to limit recognition of P(4) Arg. The effects of putative S(4) and S(6) mutations were determined by examining the cleavage by purified mutant enzymes of a series of fluorogenic substrates with systematic changes in P(4) and/or P(6). Whereas wild Kex2 exhibited little preference type for Arg at P(6), the T252D mutant and T252D/Q283E double mutant exhibited clear interactions with P(6) Arg. Moreover, the T252D and T252D/Q283E substitutions altered the influence of the P(6) residue on P(4) recognition. We infer that cross-talk between S(4) and S(6), not seen in furin, allows wild type and mutant forms of Kex2 to adapt their subsites for altered modes of recognition. This apparent plasticity may allow the subsites to rearrange their local environment to interact with different substrates in a productive manner. E255I-Kex2 exhibited significantly decreased recognition of P(4) Arg in a tetrapeptide substrate with Lys at P(1), although the general pattern of selectivity for aliphatic residues at P(4) remained unchanged.

Amino Acid Sequence

Humans

Proprotein Convertases - metabolism

Furin - chemistry

Molecular Sequence Data

Substrate Specificity

Proprotein Convertases - chemistry

Saccharomyces cerevisiae Proteins - genetics

Proprotein Convertases - genetics

Saccharomyces cerevisiae Proteins - metabolism

Protein Binding

Molecular Structure

Subtilisins - metabolism

Kinetics

Mutation

Binding Sites

Saccharomyces cerevisiae

Amino Acid Substitution

Furin - metabolism

Saccharomyces cerevisiae Proteins - chemistry

Details

Title: Subtitle: Plasticity of extended subsites facilitates divergent substrate recognition by Kex2 and furin
Creators: Laura Rozan - Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA
Damian J Krysan
Nathan C Rockwell
Robert S Fuller
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.279(34), pp.35656-35663
DOI: 10.1074/jbc.M405362200
PMID: 15159396
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Grant note: GM 39697 / NIGMS NIH HHS P30 CA46592 / NCI NIH HHS
Language: English
Date published: 08/20/2004
Academic Unit: Microbiology and Immunology; Stead Family Department of Pediatrics; Infectious Disease (Pediatrics)
Record Identifier: 9984093336302771

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